IBI Volume 6 Issue 4

Volume 6 Issue 4

Peer Reviewed

New opportunities for Nanoparticles through Nasal Delivery Designing Increasingly Complex Flow Cytometry Panels for High-Throughput Immunophenotyping Adapting to the Ever-evolving Cell and Gene Therapy Landscape The Cloud Isn’t Everything De-mystyfying Cloud Based LIMS Sponsor Company:

www.international-biopharma.com

GREAT FLUIDIC SOLUTIONS

BIOTECH LIQUID µFLOWMETER Continuous Flow Measurement 10 nl – 80 µl/min

BIOTECH LIQUID FLOWMETERS Compatible with Leading Chromatography Software

www.biotechfluidics.com Europe: Biotech AB Phone: +46 300 569 180 info@biotechfluidics.com

II INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

United States: Biotech USA LLC Phone: 612 703 5718 sales@biotechfluidics.com

Japan: BioNik Inc. Phone: +81 545 389 125 info@bionikinc.com

Winter 2023 Volume 6 Issue 4

Contents 04 Foreword TALKING POINT 06 New Opportunities for Nanoparticles Through Nasal Delivery

DIRECTOR: Mark A. Barker INTERNATIONAL MEDIA DIRECTOR: Anthony Stewart anthony@senglobalcoms.com EDITORIAL MANAGER: Beatriz Romao beatriz@senglobalcoms.com DESIGN DIRECTOR: Jana Sukenikova www.fanahshapeless.com FINANCE DEPARTMENT: Akash Sharma accounts@senglobal.co.uk RESEARCH & CIRCULATION: Jessica Chapman info@senglobalcoms.com COVER IMAGE: iStockphoto © PUBLISHED BY: Senglobal ltd.

Unit 5.02, E1 Studios, 7 Whitechapel Road, E1 1DU, United Kingdom Tel: +44 (0)20 4541 7569 Email: info@senglobalcoms.com www.international-biopharma.com All rights reserved. No part of this publication may be reproduced, duplicated, stored in any retrieval system or transmitted in any form by any means without prior written permission of the Publishers.

The next issue of IBI will be published in Spring 2024.

ISSN No.International Biopharmaceutical Industry ISSN 1755-4578.

The opinions and views expressed by the authors in this journal are not necessarily those of the Editor or the Publisher.

Please note that although care is taken in the preparation of this publication, the Editor and the Publisher are not responsible for opinions, views, and inaccuracies in the articles. Great care is taken concerning artwork supplied, but the Publisher cannot be held responsible for any loss or damage incurred. This publication is protected by copyright.

2023 Senglobal ltd.

Volume 6 Issue 4 – Winter 2023

www.international-biopharma.com

Having gathered momentum for decades, nanoparticles (NPs) reached a critical new phase in their evolution during the pandemic, with both the Pfizer/BioNTech and Moderna mRNA vaccines employing this approach to protect millions across the world against Covid-19. The growing focus on nanoparticle systems has been driven by the various benefits they offer as a carrier for therapeutic agents, and particularly biologics such as nucleic-acid-based therapies. In this interview feature, Dr. Irene Rossi and Dr. Eleanor Canipa at Nanopharm, talk about this emerging market and insights into the development process.

REGULATORY & COMPLIANCE 12 Supporting Investigators and Sites in the Evolution to Decentralised Clinical Trials The growing uptake of decentralised clinical trials in recent years reflects several key drivers such as advances in remote monitoring technology and data analytics, the need to access broader, more diverse trial populations, and demand in clinical development for cost efficiencies and more meaningful, real-world study outcomes.

If investigators and sites are partnered, supported, trained in the right way, the benefits should be felt all round. Harpreet Gill of ICON explains that we are at a point that if this approach is really to be considered business as usual, it needs to have acceptance by and support from all stakeholders, including investigators and site staff.

RESEARCH / INNOVATION / DEVELOPMENT 16 Designing Increasingly Complex Flow Cytometry Panels for High-Throughput Immunophenotyping Offering simultaneous detection of an expanding array of cell markers, flow cytometry has become an indispensable high-throughput tool, used for the detection and quantification of diverse immune cell populations within heterogeneous samples.

With a broad range of research and clinical applications, this analytical technique plays a pivotal role in understanding the immune system's role in disease progression, monitoring patient responses to treatment, and assessing the safety and efficacy of novel therapies and vaccines. Sharon Sanderson at Bio-Rad Laboratories, discusses that to ensure panel building success, a selection of best practice guidelines surrounding core aspects of immunophenotyping can be followed to assist with experiment design.

20 Regulatory Changes and Partnerships will Enable Gene Editing Technologies as a Platform-based Therapeutic Approach It has long been postulated that the genetic code containing the instructions for life could be precisely edited to correct and cure human disease. That dream began the transition towards reality with the breakthrough 2012 publication that first demonstrated use of the CRISPR-Cas9 endonuclease system. Since then, CRISPR-Cas-based genome editing technologies have changed virtually every facet of basic and applied biological research, holding the potential to push both medicine and science into an age of innovation unlike any seen previously. Jeff Briganti at Aldevron outlines the regulatory changes and partnerships will enable gene editing technologies as a platform-based therapeutic approach.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 1

Contents 24 Tackling the Complexities of Bringing Oncolytic Viruses to Market Oncolytic viruses (OVs) have emerged as a remarkable non-to-lowtoxic and non-invasive alternative to traditional cancer treatments.

These relatively new cancer therapeutics are being increasingly explored by the pharmaceutical industry to benefit from their selectivity and potential to enhance existing medicines. Despite some early clinical trial successes, numerous obstacles impede the progress of this exciting therapeutic area. In this article, Kai Lipinski, at ReciBioPharm, delves into the challenges that OV developers face in pursuing project success and assesses potential resolutions to bring pioneering new treatments one step closer to launch.

THERAPEUTICS 30 A Novel Approach to Inhalation Therapy Inhalation therapy is used to treat patients with both acute and chronic respiratory diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, and more. There is the obvious advantage of target organ delivery for afflictions of the throat, lung, and oesophagus. However, inhalation therapy can also have distinct advantages for

other conditions, although not delivered directly to the target organ. The delivery of therapies into the alveolar spaces of the lung results in the rapid absorption of oxygenated blood.

Paul Hardman at Broughton talks about the advantages and disadvantages of inhalation therapy.

MANUFACTURING & PROCESSING 34 Adapting to the Ever-evolving Cell and Gene Therapy Landscape Over the past 10 years, the cell and gene therapy (C&GT) space has grown rapidly, with the number of innovative medicines entering the development pipeline steadily rising year on year. In this article, Andelyn Biosciences examines the drivers behind current trends in the C&GT space and the obstacles developers and manufacturers face on their journey to market. Leveraging its unique insight, Andelyn Biosciences emphasizes the importance of implementing specific strategies to quickly adapt to changing C&GT needs as they emerge.

38 Adapting to Supply Chain Issues Surrounding Antibody-based Therapy Production The complexity and diversity of antibody-based therapies have been increasing with the growing understanding of the intricate mechanisms involved in immune responses, the discovery of novel therapy targets and the development of new technologies for antibody production. As a result, the biopharmaceutical industry has placed greater significance on advancements in manufacturing and collaborations within the supply chain. However, introducing new products or materials into a pre-existing process can be time-consuming and costly, as regulatory agencies must be notified and the entire manufacturing process reapproved. In this article, Aaron Moulin at Purolite, discusses the need to establish robust supplier networks that will support streamlining biologics development and manufacturing processes. He also explores some of the emerging purification challenges associated with developing novel antibodies, emphasizing the importance of implementing a quality-by-design (QbD) approach when adopting new resin technologies.

TECHNOLOGY 40 The Cloud Isn’t Everything – De-mystyfying Cloud Based LIMS Without a doubt, one of the hot topics in Laboratory informatics for a number of years has been the use of the cloud for hosting Laboratory Information Management Systems (LIMS) and other lab-based systems. However, is the cloud and whatever cloud model is used or recommended (i.e., Platform as a Service, Software as a Service, Infrastructure as a Service or whatever other four-letter acronym someone comes up with) really the be-all and end-all of lab informatics? Simon Wood at Autoscribe Informatics outlines the risks and benefits of cloud-based LIMS. 42 The Benefits of Digital and Electrochemical Lateral Flow Assays Lateral flow tests (LFTs) are simple diagnostic technologies that allow rapid and low-cost detection of a given analyte at the point of care level. While LFAs have widespread uses including environmental testing they are more commonly associated with healthcare testing, for example the pregnancy or the SARS-COV-2 Antigen test. Ben Edwards, Uroš Zupančič and Paul Ko Ferrigno at éclateral Ltd outline the benefits of digital and electrochemical lateral flow assays.

2 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Winter 2023 Volume 6 Issue 4

25 years advancing every day

Supplying what’s needed now and for what’s next The biotechnology industry is poised to deliver amazing products and solutions that will dramatically change millions of lives for the better. Aldevron is ready.

As a globally trusted CDMO, our years pioneering ground-breaking solutions to advance biologic manufacturing for research use, as well as cGMP for clinical and commercial applications, has enriched us with a body of knowledge that is critical to support these next series of breakthroughs.

Contact us to advance your program. +1 (701) 297-9256 | Toll-free (U.S. and Canada): +1 (877) 787-3362 aldevron.com/advancing-every-day

23-ALD-AED-G2B

Foreword The inhalation of pharmaceutical active substances is one of the earliest routs of administration for pharmaceutical active substances used by humans. The devices used to deliver drugs to the respiratory system or nasal cavities have been optimized over the past century to effectively deliver a wide range of drugs. Up to now these devices have shown to be effective mainly for small molecule drugs. But in the past decade we have seen different players, academic and industry, developing devices and formulations to open up this route of administration to biologics, namely to proteins, nucleic acids and viral vectors. This years Drug Delivery to the Lungs (DDL) conference again showcased efforts to make the delivery of biologics feasible. This issue of IBI features two articles highlighting new technologies and formulations that may lead to effective new drug products. Dr. Irene Rossi and Dr. Eleanor Canipa at Nanopharm explain how actives formulated as nanoparticles can be effectively delivered to the nasal cavity. On a similar topic, Dr. Nveed Chaudhary at Broughton explores different classical pharmaceutical inhalation devices and highlights what pharma my learn from the delivery devices used by the tobacco industry.

A recurring topic of this publication are articles about different aspects of Advanced Therapeutics and Medicinal Products (ATMPs) and this issue is no exception. Jeff Briganti at Aldeveron highlights the advanced in CRISPR Cas9 mediated gene editing. This technology, once available as a therapy, will enable the treatment of many debilitating illnesses directly at its genetic source. Till then strategic partnerships need to be formed to overcome technical and regulatory hurdles. Andelyn Biosciences highlights the cell and gene therapy landscape form a CDMO point of view, discussing the need to innovate production technologies, changing regulatory demands and at the same meeting cost expectations.

An exciting development in the ATMP field are so-called oncolytic viruses. These viruses are highly selective in infecting specific tumor tissues where they can disrupt tumor progression and enhance sensitivity to standard of care treatments. Kai Lipinski at ReciBioPharm gives an insight into recent clinical results and what to expect in the near future.

Complex and in part individualized investigational therapies will bring their own challenges to clinical studies as suitable patients will be scatter across the globe. This will increase the need to produce clinical trial material faster, more efficiently and cost effective as Ed Groleau at PCI Pharma Services explains. Another great change lies in the collection of data from different clinical sites and settings. Harpreet Gill at IQVA gives an insight into digital solutions on offer to monitor, record and analyze clinical data form decentralised clinical settings.

One critical aspect of clinical studies are meaning full diagnostic data to monitor the efficacy of the treatments under investigation.

Sharon Sanderson at BioRad explains how multiplexed Flow Cytometry may be used to phenotyping a patient’s immune status by monitoring the composition and status of immune cells.

Flow Cytometry is a powerful diagnostic toll, however a complex technology which is for experts only. Another diagnostic tool which we all have, unfortunately become very accustomed to during the COVID pandemic, are lateral flow assays. Benjamin Edwards; Uroš Zupančič; Paul Ko Ferrigno at éclateral Ltd explain the possibility of digital and electrochemical lateral flow test for different clinical markers and their use in a clinical or home care setting. These technologies offer a easy to use solution to monitor disease biomarkers to better manage medication and care.

Lastly, I want to thank the team at Senglobal for putting together four truly interesting and informative issues throughout this year.

I am looking forward the new innovative biopharmaceutical the year 2024 has in stall for us!

Dr. Steven A. Watt, Head of Business Development at A&M STABTEST GmbH

IBI – Editorial Advisory Board •

Ashok K. Ghone, PhD, VP, Global Services MakroCare, USA

•

Bakhyt Sarymsakova – Head of Department of International Cooperation, National Research Center of MCH, Astana, Kazakhstan

•

Jeffrey W. Sherman, Chief Medical Officer and Senior Vice President, IDM Pharma.

•

Lorna. M. Graham, BSc Hons, MSc, Director, Project Management, Worldwide Clinical Trials

•

Mark Goldberg, Chief Operating Officer, PAREXEL International Corporation

•

Maha Al-Farhan, Chair of the GCC Chapter of the ACRP

•

Rick Turner, Senior Scientific Director, Quintiles Cardiac Safety Services & Affiliate Clinical Associate Professor, University of Florida College of Pharmacy

•

Catherine Lund, Vice Chairman, OnQ Consulting

•

Cellia K. Habita, President & CEO, Arianne Corporation

•

Chris Tait, Life Science Account Manager, CHUBB Insurance Company of Europe

•

Deborah A. Komlos, Senior Medical & Regulatory Writer, Clarivate Analytics

•

Elizabeth Moench, President and CEO of Bioclinica – Patient Recruitment & Retention

•

Robert Reekie, Snr. Executive Vice President Operations, Europe, Asia-Pacific at PharmaNet Development Group

•

Francis Crawley, Executive Director of the Good Clinical Practice Alliance – Europe (GCPA) and a World Health Organisation (WHO) Expert in ethics

•

Stanley Tam, General Manager, Eurofins MEDINET (Singapore, Shanghai)

•

Hermann Schulz, MD, Founder, PresseKontext

•

Stefan Astrom, Founder and CEO of Astrom Research International HB

•

Jim James DeSantihas, Chief Executive Officer, PharmaVigilant

•

Steve Heath, Head of EMEA – Medidata Solutions, Inc

4 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Winter 2023 Volume 6 Issue 4

Recombinant Insulin for upstream processes

Enabling better medicines Enabling better lives

Grow your cells with Recombinant Insulin As the leading supplier of Recombinant Insulin for serum-free and chemically defined cell culture media, we can help you keep development on track and production flowing.

Novo Nordisk Pharmatech A/S remains the best supplier of Recombinant Insulin – beginning with the quality of our Insulin sourced directly from Novo Nordisk, the world’s largest producer.

Manufacturing and quality control, precision delivery and a risk mitigation strategy that assures continuous availability are a few reasons why you should grow your cells with our Recombinant Insulin.

Are you attending Cell 2023 in London?

Visit us at booth 16

Learn more about our Recombinant Insulin at novonordiskpharmatech.com

www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 5

Talking Point

New Opportunities for Nanoparticles Through Nasal Delivery Having gathered momentum for decades, nanoparticles (NPs) reached a critical new phase in their evolution during the pandemic, with both the Pfizer/BioNTech and Moderna mRNA vaccines employing this approach to protect millions across the world against Covid-19.

We speak to Dr. Irene Rossi and Dr. Eleanor Canipa at Nanopharm, about this emerging market and insights into the development process.

Q: What are the advantages of nanoparticle systems?

A: The growing focus on nanoparticle systems has been driven by the various benefits they offer as a carrier for therapeutic agents, and particularly biologics such as nucleic-acid-based therapies. Here, nanoparticle encapsulation provides biologics with a stable platform to cross multiple biological barriers and support effective uptake within target cells, whereas without encapsulation, some biologics can degrade quickly. Specific advantages of nanoparticle systems include their high level of encapsulation efficiency; the fact that the molecule is protected against enzymatic degradation; their inherent adjuvant properties, which result in increased immunogenicity for RNA and DNA vaccinesi; and the improved pharmacokinetic profile they can deliver by avoiding renal excretion and clearance via the mononuclear phagocyte system (MPS).

Compared with viral vectors, nanoparticle systems support a reduced immune response, mitigating against the risk of the therapy triggering an adverse effect. They offer other benefits too, including low toxicity and the potential to deliver large payloads with a controlled, sustained release.

Q: How are nanoparticle systems formulated?

A: Nanoparticles can be formulated using several methods, the foremost being to dissolve lipids (which are mixed with a nucleicacid solution) in ethanol to produce lipid nanoparticles (LNPs) via self-assembly. Alternatively, microfluidic mixing processes have been designed to achieve rapid and controlled folding of fluids within microseconds to milliseconds for precise control of particle size, more homogeneous size distribution, higher encapsulation efficiency, and greater reproducibility than bulk methods. An example of this approach is hydrodynamic flow focusing, a microfluidic laminar flow method where particles are formed at the interface of laminar flow streams. However, this process is limited in throughput (< 10mL/h) and is mainly used for preparing liposomes, which have less structural complexity than LNPs. Another rapid-mixing process is ‘Tjunction mixing’, where turbulent mixing in a macroscopic channel (> 1mm) can achieve small nanoparticle sizes (< 100nm), but this method cannot scale 6 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

down to the small volumes (μL) needed for high-throughput library screening of nanomaterials.2 For each method, particle size – and the ability to control it – is an important parameter as it greatly influences the fate of the nanoparticle in vivo. Bulk processes, namely self-assembly, do not require specialised equipment, but they lack precise control over mixing time and thus create large (> 100 nm), polydisperse particles with low encapsulation efficiency that vary from batch-to-batch and can require more downstream processing in large-scale manufacturing. This reduces yields and increases production costs.

Q: Why is there an interest in nanoparticle for intranasal drug delivery?

A: The nasal cavity is highly vascularised, providing rapid absorption and distribution of drugs, resulting in a faster onset of action compared to other routes. The avoidance of hepatic first-pass metabolism is another benefit, as drugs delivered intranasally bypass the liver initially. This approach is a non-invasive and patient-friendly method, especially for children and elderly individuals who may have difficulty with injections.1 To date, injection has been the predominant route of administration for nanoparticle formulations. However, the influence of Covid-19 has placed a greater focus on the potential for intranasal delivery of nanoparticle molecule systems as a more patient-friendly route for tackling airborne respiratory viruses and the potential for an improved or faster outcome.

Since the mucosa of the upper respiratory tract is the principal site of infection for viruses such as SARS-CoV-2 and influenza, delivering drugs intranasally can, if desired, facilitate the activation of the mucosal immune system, either as a prophylactic measure in the case of vaccines or for therapeutic treatments aimed at addressing the inflammatory response in patients infected by these viruses. Moreover, some drug products are designed to directly target either the virus itself or the receptors mediating viral cell entry, and often these are nucleic-acid-based therapies that would deploy nanoparticle systems.3 Beyond vaccines and antiviral therapies, intranasal delivery also presents a potential pathway for nanoparticle-encased drugs to treat conditions affecting the Central Nervous System (CNS) via the nose-to-brain pathway, which is primarily achieved by targeting the olfactory and turbinates regions. The nose-tobrain pathway offers a non-invasive route to deliver therapeutics directly to the brain, bypassing the blood-brain barrier. This approach shows potential for treating neurological conditions like Alzheimer's and Parkinson's disease.4 In addition, nanoparticlebased intranasal drug delivery can be combined with other innovative approaches, such as gene editing technologies like CRISPR-Cas9. This combination could pave the way for targeted Winter 2023 Volume 6 Issue 4

Talking Point gene therapies for various genetic disorders affecting the respiratory system and CNS.5 To realise the exciting potential of intranasal delivery for nanoparticles in applications such as these, however, a variety of issues must be addressed. Either in the case of reformulating an existing nanoparticle injectable or introducing a new nasal nanoparticle therapy, the process must answer some fundamental questions from the outset to accelerate and de-risk the development of the device and formulation combination.

Q: What are the essential first steps when formulating nanoparticle molecule systems for intranasal delivery?

A: At Nanopharm, our expertise in formulation science tells us that the starting point for any project of this type should in fact be to assess the characteristics of the nanoparticle system in question, which includes consideration of the active ingredient and target therapeutic effect.

While intranasal drug delivery has several advantages, there are challenges that need to be addressed. Nasal drug formulations must be carefully designed to ensure proper bioavailability and stability during storage and administration. The nasal cavity can also be sensitive to certain properties, such as pH and osmolality, which can impact tolerability and patient compliance but will also affect the NANOPARTICLE themselves. Ensuring precise control over the particle size and formulation characteristics is crucial for the efficacy and safety of nanoparticles.1 It is important to remember, after all, that in the case of nanoparticle systems, the active drug is not free in a solution, suspension or powder, but rather it is encapsulated, conjugated or complexed within a nanoparticle. As such, the focus is also on formulating the nanoparticle, not the drug itself, and protecting it on its journey to the nose, including ultimately the facilitation of deposition, release of the active ingredient and its resultant therapeutic effect. The constituent lipids or polymers that make up the nanoparticle will demonstrate individual characteristics related to particle size, structure, deposition, mucoadhesion, and stability as well as pharmacokinetics in terms of absorption, distribution, metabolism, and excretion (ADME).

The potential for agglomeration or bursting, for example, must be understood and taken into consideration when evolving the formulation for intranasal delivery, particularly since there are limits to what the nose can tolerate with regards to characteristics such as pH and osmolality. Modulating these properties must be achieved in such a way that it does not affect the stability of the nanoparticle, which may mean that some pH ranges are not compatible. Carefully chosen tonicity adjusting agents, tested for compatibility, may, along with surfactants or other elements, be needed to stabilise the system and avoid agglomeration or release of the nanoparticle’s payload.

By accurately establishing the physico-chemical properties at play, the variables within the formulation process can be managed and their potential to adversely impact stability can be limited.

As intranasal drug delivery using nanoparticles continues to gain interest, regulatory agencies will play a crucial role in www.international-biopharma.com

evaluating safety and efficacy. Guidelines specific to intranasal delivery and nanomedicine products may need to be developed to address unique considerations.1 Q: What are some of the key considerations when reformulating an injectable nanoparticle system to a nasal nanoparticle system?

A: The sterility requirements from regulators of the original injectable formulation means that preservatives and other excipients are not often considered before the reformulation process. However, sterile manufacturing for intranasal systems is less well-established – aside from the careful consideration for the formulation process itself, particular attention should be paid to incorporating specific excipients for viscosity control, which may not be compatible with elements of the current injectable sterile production; and the more specialised capabilities of assembling nasal devices may limit this option further. Therefore, preservatives may be required for intranasal systems to prevent contamination and enable an acceptable shelf life – particularly if multiple doses are required – the presence of preservatives can add greater complexity to early formulation development, even if it simplifies the final manufacturing process, because their addition could influence the stability of the NANOPARTICLE − either positively or negatively. Preservatives, therefore, need to be profiled accurately and their interaction with other formulation constituents monitored closely, as per the Nanopharm strategy for development.

For example, there is a risk that the preservative may enter the nanoparticle system, rendering it ineffective since it is no longer able to “preserve” the aqueous medium of the bulk formulation.

This might also disrupt the nanoparticle structure and create the potential for the encapsulated drug substance to be released.6 There are, however, formulation strategies that can be adopted to counter this particular risk.

In addition, preservatives may also encourage aggregation of the lipid systems, again disrupting integrity and risking the premature release of the drug substance or affecting stability of the final nasal product and inducing undesirable immune reactions. However, there is also evidence that, in the right circumstances and at the right concentrations, some preservatives − including Benzalkonium Chloride, the most commonly used preservative in liquid multi-dose nasal sprays – can enhance the stability of LNPs.7 So, clearly, there is not a one-size-fits-all approach, and performing adequate pre-formulation studies is critical to avoid issues later in the development process.

Adjuvants are another area of consideration for nasal nanoparticle formulations targeting vaccination. Their inclusion supports a rapid and prolonged immune response within the mucosal tissues but, again, it is essential to understand their wider influence on the stability of the nanoparticle system and the properties of the final nasal formulation through a managed testing and analysis programme. Furthermore, it is important to highlight that nanoparticles themselves can act as an adjuvant and this attribute can be amplified if the right components are employed in their construction. In other cases, adjuvants can be associated to the nanoparticle structure to augment the immune response.8 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 7

Talking Point Q: Which is the preferred form for intranasal nanoparticle delivery: liquid or powder?

A: Numerous factors will contribute towards the decision about whether a nanoparticle-based vaccine or therapy should be optimised as either a liquid or a dry powder. Powder formulations can overcome the stability concerns inherent with liquid formulations, while also offering the potential for an enhanced immune response through prolonged exposure on the nasal mucosa. By avoiding the need to be handled via cold-chain logistics, powder formulations can also reduce costs and complexity, creating opportunities for cost-effective vaccination programmes in territories where transportation and storage infrastructures are restricted.

Furthermore, powder formulations are preferable to liquid formulations when the delivery of a high drug payload is required.

Doses that require a high volume of liquid can cause tolerability issues for patients, although there are a variety of different methods that can be explored to overcome this issue and increase the amount of active ingredient reaching the target area, such as higher drug concentrations or methods to enhance permeation and absorption in the nasal cavity.

However, powder systems are generally more complex and expensive to develop and manufacture, especially if reformulating from an existing liquid-based injectable formulation. Therefore, if the aqueous system can be kept stable then this is often the quickest and most cost-effective option, and the preferred starting point, particularly for early-stage clinical studies – even if the final commercial format might bridge over to a dry powder product.

Q: What are the different methods to manufacture nanoparticle powder formulations for nasal delivery?

A: There are various routes to achieving a dry powder formulation, with freeze drying and spray drying the primary options for biologic molecules. Spray drying, which involves the rapid evaporation of a solution or suspension at high temperatures, can present challenges, however. Given the relatively low melting point of the lipids that constitute nanoparticle systems and the drying process involved, there is a risk of compromising the integrity of the nanoparticles and, therefore, the bioavailability and efficacy of the resulting powder formulation.

These problems can often be avoided through freeze drying.

This is a more commonplace lyophilization process that involves the freezing of the product prior to moisture being removed by drying and sublimation. Freeze drying, however, can only be achieved within a critical temperature range and any variance can impact on the stability of the nanoparticle. This requires the freezing conditions as well as the primary and secondary drying conditions to be defined based on the characteristics of the nanoparticle system, with the formulation composition screened to ensure nanoparticle structure and size are maintained in the powder cake obtained. This would be achieved using techniques such as transmission electron microscopy (TEM), x-ray microscopy and dynamic light scattering (DLS).

Moreover, freeze drying is not suitable to directly obtain the form of powders required for nasal delivery and a second 8 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

process, such as sieving, blending, mixing and/or milling, would be required to obtain a final product that can be delivered by nasal spray and deposit in the target region of the nasal cavity. Further drawbacks to freeze drying include the energy consumption levels required during the process, the comparatively slow rate of production, and the relatively low yields, which can impact on overall cost.

To assess whether the nanoparticle and overall formulation would be suited to either spray drying or freeze drying, close analysis is carried out via modulated differential scanning calorimetry (DSC) or freeze-drying microscopy. This elicits data on the physico-chemical characteristics of the nanoparticle suspension, providing the means to analyse a particular product to ensure its integrity is maintained in the creation of a powder state. If the results subsequently highlight a need to modify the formulation’s properties to facilitate a given process − such as increasing or decreasing the boiling or freezing point − components can be added to the formulation. Sugars and polyalcohols, for example, are recognised as cryoprotectants, stabilizers or bulking agents.

Q: What assessment can be made of the performance of the final, intranasally delivered nanoparticle formulation?

A: Nasal formulations are usually screened using nasal devices following a design-of-experiment approach, where typical excipients used for nasal delivery, including preservatives, mucoadhesive polymers, absorption enhancers and buffers, are tested for their influence on properties such as rheological behaviour, droplet-size distribution (DSD- an example in Figure 1), spray pattern (SP), plume geometry (PG), and spray-content uniformity (SCU). The outputs from these experiments allow the optimal formulation composition to be defined as well as enabling product performance to be predicted in relation to the design space investigated.

Formulation development of a nanoparticle for nasal delivery starts from the characterization of the nanoparticle system itself to define properties such as size (by dynamic light scattering), interfacial charge between the nanoparticle aqueous media (zeta-potential), encapsulation efficiency and stability. These properties may change through the addition of other formulation enhancers that may be required, which makes it essential to monitor and modulate them from the start to ensure there are no surprises later and that the impact of the formulation design on critical attributes is consistently understood.

To do this, stressful storage conditions may need to be defined to be able to select the optimal variants for formulation optimisation and clinical trials.

In the case of nanoparticles, it is fundamental to determine the integrity of the system after formulation development and after spraying from the nasal device. This is because the mechanisms involved in forming droplets within the nasal spray pumps impart high shear stresses (albeit for a very short timeframe) which may disrupt the nanoparticle structure. This can be assessed with techniques like dynamic light scattering (DLS – an example of size assessment as per Figure 2) and turbidity measurements, which help identify precipitation or any change in size due to the aerosolisation process. This analysis can be conducted Winter 2023 Volume 6 Issue 4

Talking Point

Figure 1. LNP DSD data using Aptar Pharma’s Classic ‘CPS’ nasal pump

together with specific methods for quantification (for example UV-Visible photometry, fluorescence, polymerase chain reaction or gel electrophoresis) and in vitro evaluation of the activity of the biological entity delivered by the system, such as mRNA transfection.

Q: What other considerations are there?

A: Generally speaking, nanoparticles reformulated from injectable to nasal delivery have good cell permeability, but mucoadhesive polymers may need to be added to prolong retention. It follows that the link between payload and delivery efficiency may then need to be assessed as the dose usually administered via injection has to be adjusted based on this new method of administration.

To help with this, in silico tools, such as physiologically based pharmacokinetic (PBPK) modelling, can be used to drive formulation development.

To do this, an understanding of where in the nasal cavity the drug will be deposited is necessary, since each region has a different physiology and this in turn impacts residence time and permeation pathways. To assess this in vitro, an anatomically accurate and clinically validated nasal cast can be employed to assess where the drug will be deposited after actuation from the device (Figure 3). This information, together with pre-existing in vivo data (in the case of the re-formulation of injectable nanoparticles to nasal delivery) or preclinical in vivo data (for new molecular entities), can be used to build a PBPK model able to predict the pharmacokinetic properties of the nasal product once delivered into the nose of humans, de-risking clinical studies.

So in Summary..?

Nanoparticle systems present significant potential for the delivery of new modalities to the nose. Nasal drug delivery

Figure 2. DLS before and after spraying from Aptar Pharma’s CPS nasal pump www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 9

Talking Point

Figure 3. AERONOSE® Nasal Cast, co-developed and co-owned by Aptar Pharma, Diffusion Technique Française and the French University of Tours for analysis of nasal drug deposition

using nanoparticles holds great promise for treating not only respiratory infections but also various Central Nervous System (CNS) disorders. However, reformulating a legacy injectable system requires careful evaluation and adaptation to successfully transition to this alternative route of administration.

Pre-formulation studies and targeted in vitro and in silico testing − focused on assessing the nasal product performance and nanoparticle system integrity when it reaches the site of action − are fundamental to define the best formulation design space, which will accelerate and de-risk product development and be instrumental in realising the full potential of nanoparticles in intranasal drug delivery.

For enquiries regarding formulation development and device characterisation, contact Nanopharm, an Aptar Pharma company at info@nanopharm.co.uk or see more of their services at nanopharm.co.uk.

REFERENCES 1.

2.

3.

4.

5.

6.

7.

8.

L.-A. Keller, O. Merkel, A. Popp, Drug Deliv. Transl. Res., 2022 Apr;12(4), 735-757 S. J. Shepherd, C. C. Warzecha, S. Yadavali, R. El-Mayta, M.-G. Alameh, L. Wang, D. Weissman, J. M. Wilson, D. Issadore, M. J. Mitchell, Nano Letters 2021 21 (13), 5671-5680 C. Rey Blanes, R. Beaird, M. Buhecha1, E. Axioti, W. Terrell1, N. Karavas, M. Isreb, R. Price, J. Shur, I. Rossi, Drug Delivery to the Lungs, (33), 2022 S. J. Shepherd, C. C. Warzecha, S. Yadavali, R. El-Mayta, M.-G. Alameh, L. Wang, D. Weissman, J. M. Wilson, D. Issadore, M. J. Mitchell, Nano Letters 2021 21 (13), 5671-5680 K. Paunovska, D. Loughrey, J. E. Dahlman, Nature Reviews Genetics 2022, (23), 265–280 D. Watrobska–Swietlikowska AAPS PharmSciTech, 2020, 21, 7-10 K. Nakamori, T. Nakajima, M. Odawara, I. Koyama, M. Nemoto, T. Yoshida, H. Ohshima, K. Inoue, Chem Pharm Bull (Tokyo) 1993 Jul;41(7),1279-1283 N. K. Childers, K. L. Miller, G. Tong, J. Carlos Llarena, T. Greenway, J. Terry Ulrich, S. M. Michalek ASM Journals, Infection and Immunity, 1 October 2000, 10 (68)

10 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Dr. Irene Rossi Irene Rossi is a Senior Specialist in Formulation Development at Nanopharm, an Aptar Pharma Company (UK). Irene obtained her Pharmacy MSc in 2014 and a PhD in Drugs, Biomolecules and Health Products at the University of Parma (Italy) in 2019. She joined Nanopharm in the same year leading a group focused on the development of new formulation technologies for OINDP products comprising both small molecules and new modalities. Up to date, Irene has published and submitted 7 original papers and 2 patent applications on the development of DPI for pulmonary vaccination and on the formulation of low global warming pMDIs. She has presented her work at more than 20 international conferences.

Dr. Eleanor Canipa Eleanor is a chartered chemist and currently works as the Senior Business Development Manager at Nanopharm, an Aptar Pharma company, with a long track record in the medical and pharmaceutical industries. With a degree in Business Management and Marketing from the Open University, a Ph.D. in the Chemistry of Au and Pd Nanoparticles from the University of York and an active committee member of the Royal Society of Chemistry’s Marketing Group, Eleanor's experience includes bespoke analytical development and validation to cGMP, expanding capabilities in various test houses, and facilitating cross-departmental collaboration. Eleanor is particularly passionate about inhalation devices and now specialises in Nasal Drug Delivery (SNDD) for Central Nervous System (CNS) drug delivery solutions, where she continues to make impactful strides within Nanopharm.

Winter 2023 Volume 6 Issue 4

dƌƵĞYƵĂŶƚ ^ŵĂůů ZE ^ĞƋ <ŝƚ <ĞLJ ĞŶĞĨŝƚƐ • &ĂƐƚ͕ ƐŝŶŐůĞͲƚƵďĞ ƉƌŽƚŽĐŽů • 'ĞůͲĨƌĞĞ • SŝŶŐůĞ ĐĞůůƐ • hŶŝƋƵĞ DŽůĞĐƵůĂƌ /ĚĞŶƚŝĨŝĞƌƐ • KƉƚŝŵĂů ĨŽƌ sƐ͖ ƉůĂƐŵĂ Žƌ ƵƌŝŶĞ ƐĂŵƉůĞƐ • ,ŝŐŚĞƐƚ ĚŝǀĞƌƐŝƚLJ ŽĨ ĚŝĨĨĞƌĞŶƚ ZE Ɛ

'ĞŶyWƌŽ 'ŵď, ůƚĞŶŚƂĨĞƌĂůůĞĞ ϯ ϲϬϰϯϴ &ƌĂŶŬĨƵƌƚ Ăŵ DĂŝŶ www.international-biopharma.com

ǁǁǁ͘ŐĞŶdžƉƌŽ͘ŶĞƚ

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 11

Regulatory & Compliance

Supporting Investigators and Sites in the Evolution to Decentralised Clinical Trials The growing uptake of decentralised clinical trials in recent years reflects several key drivers such as advances in remote monitoring technology and data analytics, the need to access broader, more diverse trial populations, and demand in clinical development for cost efficiencies and more meaningful, real-world study outcomes.

However, we are at a point that if this approach is really to be considered business as usual, it needs to have acceptance by and support from all stakeholders, including investigators and site staff.

Historically, the physical interface between sites and study participants was the primary connection in clinical trials. While decentralised clinical trials may ease patient burden and potentially some administrative burden on sites, they can also raise concerns about loss of direct patient contact and revenue generation.

It is therefore critical that sponsors communicate effectively with their sites about how associated services, such as in-home services, will work to the advantage of not just the patient but the investigators and site staff also. Sponsors should also ensure they provide the right support and appropriate training to ensure that these advantages are recognised and exploited at site level.

A few years ago, ICON conducted a survey to get a pulse check on site sentiment and their experiences during the COVID-19 pandemic, when decentralisation really took off. At that time, the survey found that nearly 70% of site staff had little or no experience of remote processes before the pandemic.

Nonetheless, more than 78% of staff felt remote processes were to their benefit, while over 90% were expecting to see more decentralisation. It showed that there is an understanding and an acceptance that decentralisation of clinical trials is here to stay and will only grow.

The varying levels of experience and capability that sites have in managing decentralisation depends to some extent on the health care system they operate within. Across the board, exposure to related processes and technologies increased under pandemic conditions, but exposure and being equipped with the skills to use such decentralised technologies are two different things. The onus is firmly on sponsors and their partners to ensure that sites still feel comfortable with the changes and bring them on the journey.

Shifting Administrative Burden and Workload from the Site One potential benefit of decentralised clinical trials is a reduction in administrative workload for trial sites. For example, recruitment presents a significant workload challenge for investigators as well as trial sponsors. Digital patient 12 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

recruitment strategies can reduce burden from sites and support reduction of overall timelines.

As patient assessments are increasingly conducted through digital health technologies capable of primary data capture, less time is needed to interact with clinical research associates (CRAs) on clinical monitoring. In a decentralised clinical trial setting, much of this activity is automated and digitalised.

However, all these changes represent a shift in the well-established operational and business model for the site. Inevitably, these changes present new challenges for investigators and sites, such as familiarising themselves with new technology and managing the new elements of a decentralised clinical trial and, it may not necessarily feel like it’s less workload initially. This is where sponsors need to go the extra mile and look to provide a range of interventions to help sites manage the decentralised model.

One suggested tactic is arranging consultations with sites at the start of the trial, so the sites know exactly what to expect from decentralised study designs. It is also important that sites can form a comfortable relationship with service vendors in this context.

Moreover, ensuring the site understands the whole patient journey make sure the site understands the whole patient journey within the framework of trial delivery, especially the site’s facilitating that journey is vital. Additional support services, whether they address recruitment, reimbursement, or logistics, can help clarify these areas while ensuring that the administrative burden of the trial is truly removed from the site as well as the patient.

Building Confidence in the Model Decentralising a clinical trial involves innovative approaches and the sheer range of technologies now available from multiple vendors and multiple platforms can also be intimidating.

Investigators and sites not only need to understand how these technologies work, but they must also be confident they will work well, without complications such as data loss or incompatibility with data privacy regulations. Sites also want to avoid being burdened with having to provide a technology support service to patients. A busy site coordinator doesn’t want to have to start showing patients how to log on to a system or how to correct their e-diary.

Moreover, sites need reassurance that vendors such as home-health nurses are properly qualified, trained, and compliant with Good Clinical Practice (GCP) and International Conference on Harmonisation (ICH) regulations. Site staff can help to address these concerns by providing feedback on their experiences to inform continuous improvement of decentralised processes.

Winter 2023 Volume 6 Issue 4

www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 13

Regulatory & Compliance

There are also some key relationship changes to take on board. With traditional clinical trials, the main interaction is with investigator site and patient. That changes with a fully decentralised trial, where it may be a concierge support service engaging regularly patients to support them through the clinical trial journey, making sure assessments or patient surveys are completed correctly and on time, hence assuring patient compliance.

At the same time, the trial site remains responsible for medical oversight of participating patients, whether visits are site- or home-based. Although the site’s relationship with the patient is going to be different, it is important that sites do not feel they are losing touch with patients and are reassured that trial participants will be carefully looked after and supported.

For their part, patients must understand, through the trial consent process, who will guide them through the trial, and that any concerns must still be taken to the investigator responsible for medical oversight.

Direct to Patient Support Services Sponsors should seriously consider building in concierge services to support both the site and the patient. These services will need to be customised and will vary according to the characteristics of the study and the patient schedule of visits, and then integrated into the clinical trial process seamlessly so that it effectively lightens the administrative burden on trial sites. Concierge services that can provide both technical and clinical support can help with access to portals, apps and wearables and sensors but also support the drug accountability process when direct to patient shipments are used. These services are provided efficiently and remotely, and ultimately reduce unnecessary calls to sites staff. However, these professionals are also trained to triage calls and ensure the site is made aware of any medical issues to maintain the necessary medical oversight.

Additionally, sites are encouraged to build relationships with in-home health professionals, so they can be confident their activities (such as drug administration, taking blood samples, questionnaires, and clinical assessments for vital signs) are in 14 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

line with protocols while prioritising patient convenience and safety.

Decentralised Trials are a Long-term Reality If investigators and sites are partnered, supported, trained in the right way, the benefits should be felt all round: faster, broader, more diverse patient recruitment; more seamless, integrated data exchange; improved granularity and real-world significance of the data collected; and reduced administrative burdens on sites.

Without the unnecessary distraction of time-consuming activities that can be automated, digitalised, and potentially managed remotely, sites can continue to pursue their research interests and care for patient needs, without diverging from the common goal of delivering timely and effective treatments.

Harpreet Gill Harpreet Gill has over 20 years of experience in clinical research. She currently leads the global project management team in the delivery of Real World Solutions to pharma, biotech and medical device organisations. Prior to this she was responsible for driving strategy and operational delivery for decentralised clinical trials, accelerating clinical trial timelines and bringing treatments to patients faster. Harpreet previously held a number of roles with increasing responsibility at IQVIA. Most recently, she led the European real world evidence project management team with responsibility for P&L and client management. She also led the global epidemiology team for some time while supporting data management, biostatistics and HEOR to align business practice and embed rigour in business review processes and project oversight. When Harpreet joined IQVIA in 2001 she successfully set-up and developed the project management office, focusing on bringing best practice project management, skills training and systems process improvement to clinical trials teams and more broadly across the organisation. Harpreet is a Chemistry graduate with a BSc(hons) from the University of North London.

Winter 2023 Volume 6 Issue 4

y archive

Compliant

Secure

Dedicated

Independent Contact us today to find out more 01933 357953 | archives@qualogy.co.uk | www.qualogy.co.uk INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 15 www.international-biopharma.com The Archivist, Qualogy Ltd, Po Box 6255, Thrapston, Northamptonshire, NN14 4ZL

Research / Innovation / Development

PEER REVIEWED

Designing Increasingly Complex Flow Cytometry Panels for High-Throughput Immunophenotyping Offering simultaneous detection of an expanding array of cell markers, flow cytometry has become an indispensable high-throughput tool, used for the detection and quantification of diverse immune cell populations within heterogeneous samples. With a broad range of research and clinical applications, this analytical technique plays a pivotal role in understanding the immune system's role in disease progression, monitoring patient responses to treatment, and assessing the safety and efficacy of novel therapies and vaccines. Careful panel design is crucial to ensuring data accuracy, particularly when performing complex multiparameter assays.1,2 To ensure panel building success, a selection of best practice guidelines surrounding core aspects of immunophenotyping can be followed to assist with experiment design.

General Sample Preparation Tips To ensure the success of your flow cytometry experiments, it's essential to prioritise meticulous sample preparation. Investing extra time in this phase can yield significantly improved results, saving you from the frustration of failed experiments and the need for multiple repetitions to achieve publication-quality data.3 Select Appropriate Tubes: Choosing the right tubes for your flow cytometry experiments is crucial. Some cytometer models allow flexibility in tube selection, like Bio-Rad’s ZE5 Cell Analyzer, but others have restrictions. It's important to use tubes that are compatible with your cytometer model, considering factors such as round or flat bottoms and the type of plastics used.

Additionally, be mindful of cell adhesion to different plastic materials. For instance, certain cell types, like monocytes, may adhere more strongly to polystyrene than polypropylene.

Selecting the appropriate plastic can help minimise cell loss and the need for harsh detachment methods.

Handle Samples Gently: Maintaining the optimal condition of your cells is paramount. Avoid subjecting them to unnecessarily harsh conditions that can lead to cell death or generate artifacts,

as this can significantly impact your results. When centrifuging samples, be cautious not to spin them at higher speeds than necessary, and avoid leaving samples in the centrifuge for extended periods, as prolonged pelleting can be detrimental.

It's advisable to handle samples gently to prevent the formation of bubbles, which can harm cells. When aspirating media, ensure that you leave some supernatant behind to prevent cells from drying out in a pellet.

Prevent Clumping: Flow cytometry relies on analysing individual, single cells. Clumps of cells can obstruct the flow cytometer, causing clogs and inconvenience to other users. To reduce the risk of clumping, consider keeping wash and media buffers at a temperature of 4°C, particularly if your experiment permits this.

Dissociate Tissues Thoughtfully: Achieving single, intact cells suitable for flow cytometry is essential. When dissociating tissues, take care to minimise cell damage. Consider methods such as tissue mincing, mesh filtering, or enzymatic digestion based on the specific requirements of your cell type. If you are working with rare cell types, opt for gentler methods to preserve them. For example, neutral protease (dispase) is a milder treatment than trypsin and is commonly used for isolating iPSCs. Some cell types, like F4/80+ macrophages and follicular dendritic cells, may require enzymatic treatment for release, and the specific enzymes needed should be determined based on established protocols.

Key Considerations for Successful Panel Design Immunophenotyping demands careful panel design as the inclusion of each additional fluorophore into the panel introduces complexities that can potentially compromise data accuracy. This is primarily due to the phenomenon of fluorescence spillover, where the emission from one fluorophore inadvertently spills over into the detection range of another, diminishing resolution and overall data quality (Figure 1).

Get to Know Your Flow Cytometer Understanding the configuration of your flow cytometer is fundamental to effective panel design.1 Most modern cytometers come equipped with three or more lasers, each

Figure 1: The electromagnetic spectrum 16 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Winter 2023 Volume 6 Issue 4

Research / Innovation / Development offering a distinct range of wavelengths. Additionally, the configuration of your cytometer hinges on the number and specifications of light filters and detectors. Together, these components determine the maximum number of fluorescent parameters that can be assessed concurrently, as well as which fluorophores can be effectively employed.

Taking a closer look at the detectors associated with each laser and the filters linked to each detector is imperative.

These filters are typically labelled as XXX/YY, where XXX denotes the median wavelength of light passing through the filter (measured in nanometers), and YY signifies the wavelength range encompassed by the filter. For instance, a filter marked as 525/50 allows light with wavelengths between 500nm and 550nm to pass through. Alternatively, filters may be designated as XXX/LP or XXX/SP, with LP indicating "Long Pass", allowing longer wavelengths than XXX, and SP indicating "Short Pass", permitting shorter wavelengths. Careful selection of fluorophores relies on matching their optimal excitation and emission wavelengths to the available lasers and filters on your detector array. While exact matches may not always be feasible, employing a spectral viewer can help determine the most likely laser/filter/fluorophore combinations that will work effectively.

Considerations when Choosing the Right Fluorophore Combination When choosing fluorophores, it is crucial to consider their individual properties to maximise data quality.4 The compatibility of fluorophores depends on their excitation and emission profiles, as well as their relative brightness. These factors collectively determine the extent of spectral overlap between fluorophores which occurs when the emission spectra of two fluorophores intersect, allowing light from one to spillover into the detection range of the other. Addressing

spectral overlap in multicolour panels is achieved through a process known as "fluorescence compensation" which can be determined using compensation controls, taking into consideration the unique fluorescent properties of each fluorophore. This method ensures that the fluorescence detected in a specific detector is attributed to the intended fluorophore (Figure 2).

Selecting fluorophores with narrow excitation and emission profiles minimises the potential for spillover. For single-laser excitation, spillover can be reduced by employing fluorophores with substantial differences in their Stokes shift, measuring the discrepancy between a dye's maximal excitation and emission wavelengths. In cases where multiple lasers are available, choosing fluorophores with significant differences in their excitation wavelengths – making them excitable by one laser but not another – significantly reduces spillover, particularly in instruments with spatially separated lasers.

To ensure reproducible results, it's imperative to use fluorophores conjugated to well-validated flow antibody clones and manufactured using robust methods to minimise lot-to-lot variation. Additionally, selecting photobleachingresistant fluorophores that remain highly stable, even during fixation, is crucial for maintaining consistent staining. Opting for fluorophores that integrate seamlessly into common experimental protocols without the need for specialised staining buffers offers cost- and time-effective advantages.

Factoring in antigen density and marker expression profiles during fluorophore selection can further mitigate spillover.

Bright fluorophores with high stain indices are ideal for detecting rare cell subsets or markers with low expression.

Conversely, dimmer fluorophores are better suited for markers with abundant expression levels. Minimising the effects of

Figure 2: Fluorescence compensation corrects for spectral overlap. Peripheral blood was singly stained with CD4 FITC, CD19 PE, or both CD4 FITC and CD19 PE. When compensation was not applied, fluorescence spillover can be seen (top panel), which is removed after compensation (bottom panel). www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 17

Research / Innovation / Development spillover and spread can also be achieved by using closely overlapping fluorophores to detect mutually exclusive markers, such as CD3 and CD19. For cell subset markers found on a common cell type or markers exhibiting continuous expression patterns, selecting fluorophores with minimal or no overlap is crucial.

and fluorophore of the primary antibody. Purchasing both the isotype control and primary antibody from the same supplier is advisable due to potential variations in fluorophore conjugation between suppliers. While the use of isotype controls can be a subject of debate among researchers, they can reveal issues such as inadequate Fc blocking.

Select Appropriate Controls Incorporating appropriate controls into flow cytometry experiments is vital for obtaining accurate, reliable data.5,6 These controls can distinguish between specific signals and background noise, ensuring the quality and validity of your findings.

Fluorescence Minus One (FMO) Controls: FMO controls help identify fluorescence spread from neighbouring channels, helping set gating parameters. Each FMO control includes all fluorescently labelled antibodies except one, allowing researchers to assess the influence of each fluorophore on the panel and its potential spread into neighbouring channels.

These controls are essential when working with multicolour panels, ensuring correct data interpretation.

Unstained Controls: These controls involve setting up the instrument using unstained cells to visualise all cells on forward and side scatter plots, thereby adjusting photomultiplier tube (PMT) voltages to distinguish cells from electronic noise.

This process helps determine background fluorescence or autofluorescence, enabling proper voltage settings for fluorescence channels. Using this approach can save time and sample during future experiments.

Single Staining and Compensation Controls: Compensation controls involve single-staining samples for each antibody in a multicolour experiment to determine spillover of fluorescence between detectors. Proper compensation settings ensure that only specific signals are used for analysis, improving data accuracy.

Fc Block Controls: The presence of Fc receptors on specific immune cells, such as monocytes, macrophages, dendritic cells, and B cells, introduces the potential for false positives and reduced resolution due to multiple antibodies binding to unintended targets. To combat this issue, Fc blocking reagents, can be added to the staining protocol (Figure 3). These reagents prevent nonspecific binding by ensuring that only antigenspecific interactions are observed. Alternatively, diluted serum from the sample's host species can be used to achieve a similar effect, e.g., using mouse serum for mouse cells.

Intracellular Staining Controls: Intracellular staining often presents challenges due to higher background levels caused by protein interactions. Isotype controls may not be suitable for this purpose, so other controls like a negative cell line or secondary antibody alone can be used to determine specific binding.

Biological Controls: Biological controls, encompassing known negative and positive samples, are essential for determining staining specificity and experiment limitations. These controls provide a reference point for interpreting results and assessing the dynamic range of fluorescence staining. Examples include cells with known antigen expression profiles, gene-edited cells, and stimulated/unstimulated controls for activation studies.

Perform Antibody Titration for Optimal Staining: Striking the right balance with antibody concentration is paramount. Excess antibodies can introduce background noise. The stain index, calculated as the difference between the median fluorescent intensity of the positive and negative populations divided by two times the standard deviation of the negative population, serves as a guiding metric (Figure 4).

Isotype Controls: Isotype controls play a critical role in surface staining experiments by verifying the specificity of antibody binding. These controls are raised against antigens not present on the cells under investigation, ensuring that the observed staining results from specific antibody interactions rather than artifacts. To maximise their effectiveness, isotype controls should match the host species, Ig subclass, concentration,

Figure 4: Calculating the stain index can help determine the ideal concentrations that will generate specific staining with the least amount of background (represented in the green box)

Figure 3: Staining of THP-1 cells with a Mouse Anti-Human CD11a antibody (blue histogram) or isotype control (red histogram) in the presence or absence of Fc block.

18 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Create Dump Channels When specific cell populations are irrelevant to the experimental design, creating a dump channel is essential.

This approach proves particularly valuable when investigating rare populations, such as stem cells, or in contexts like vaccine development, where immune system cells of interest constitute a minute fraction of the total T cell population. By employing fluorophore-conjugated antibodies specific to cells you wish to exclude but not expressed on your target cells, you can Winter 2023 Volume 6 Issue 4

Research / Innovation / Development channel can exclude dead cells when using a viability stain, reducing signal noise caused by non-specific staining and enhancing data quality.

Incorporating Best Practices with Online Panel Building Tools Leveraging online tools can simplify the complex panel design process and reduce the risk of selecting reagents that are incompatible with your flow cytometer. For example, dedicated panel building websites take instrument configuration and capabilities into account. Other online tools, such as spectral viewers, provide insights into spillover and excitation by each laser, highlighting compatibility issues between fluorophores.

Similarly relative brightness tables assist in pairing fluorophores with specific targets, while marker expression data aids in understanding expression patterns before selecting a fluorophore.

Conclusion Working with complex, multiparameter panels requires careful consideration of instrument capabilities, fluorophore properties, sample availability and antibody functionality.

Moreover, leveraging online tools has also been underscored as a valuable resource to simplify the intricate process of panel design. While the process may involve multiple design and optimisation steps, adhering to best-practice guidelines and selecting appropriate controls can make the process more efficient and cost-effective which can ultimately lead to reproducible and reliable experimental outcomes.7 REFERENCES 1.

2.

3.

4.

5.

6.

7.

Maecker HT, Trotter J (2006) Flow Cytometry Controls, Instrument Setup, and the Determination of Positivity. Cytometry A. 69(9):1037– 1042 Key Factors to Consider When Designing High-Throughput Immunophenotyping Panels, https://www.bio-rad.com/en-uk/ applications-technologies/building-optimizing-complex-highthroughput-immunophenotyping-panels?ID=b421d344-5818d8b8-f446-05f6621ae433 Set Yourself up for Success with Flow Cytometry, https://www. bio-rad-antibodies.com/blog/set-yourself-up-for-success-withflow-cytometry.html Ferrer-Font L, Pellefigues C, Mayer JU, Small SJ, Jaimes MC, Price KM. Panel Design and Optimization for High-Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry (2020) Curr Protoc Cytom. 92(1):e70. doi:10.1002/cpcy.70 Controls in Flow Cytometry, https://www.bio-rad-antibodies.com/ flow-cytometry-controls.html 5 Steps for Successful Flow Cytometry, https://www.bio-radantibodies.com/blog/5-steps-successful-flow-cytometry-results. html Optimise your Flow Cytometry, https://www.bio-rad-antibodies. com/optimising-flow-cytometry-experiments-webinar.html

Sharon Sanderson

eliminate unwanted binding, fluorescence spillover, and signal contamination from your analysis. In scenarios where the detection of rare cells, such as hematopoietic stem cells, is paramount, employing a specific fluorophore to stain all other cells ensures they are disregarded. Additionally, a dump www.international-biopharma.com

Sharon Sanderson, PhD, is a Flow Cytometry Applications Scientist and Product Manager for flow reagents. Her PhD was in cell biology focusing on cell signaling pathways in angiogenesis. Prior to joining Bio-Rad she held post-doctoral positions at The University of Oxford. Initially continuing to study the field of angiogenesis before more recent positions in immunology research.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 19

Research / Innovation / Development

PEER REVIEWED

Regulatory Changes and Partnerships will Enable Gene Editing Technologies as a Platform-based Therapeutic Approach CRISPR Cas9-mediated Genomic Editing It has long been postulated that the genetic code containing the instructions for life could be precisely edited to correct and cure human disease. That dream began the transition towards reality with the breakthrough 2012 publication that first demonstrated use of the CRISPR-Cas9 endonuclease system.1 Since then, CRISPR-Cas-based genome editing technologies have changed virtually every facet of basic and applied biological research, holding the potential to push both medicine and science into an age of innovation unlike any seen previously.

The CRISPR-Cas9 effector nuclease, from a class 2 bacterial CRISPR system, consists of a small guide RNA (sgRNA) and the Cas endonuclease.1,2 By modifying and controlling the nucleotide sequence of the guide RNA, the artificial Cas9 system could be programmed to target virtually any DNA sequence. Thus, by utilising the CRISPR-Cas9 system, delivered to a host cell either through viral or nonviral mechanisms, distinct insertions, deletions, or point mutations can be introduced at any loci of interest in the host genome through DNA repair mechanisms.3 The Clinical Promise of CRISPR-Cas9 Based Gene Editing Given the power and utility of CRISPR-Cas9-based genomic editing, it has been utilised extensively in basic as well as translational research to better understand gene function and to ultimately explore clinical targets. Studies utilising both in vitro as well as in vivo experimental models have generated compelling and promising results against a vast list of target disease indications.

In the context of cancer, CRISPR-mediated knockouts of genes essential for regulation of the cell cycle and drug resistance have been performed to modulate disease.4,5 In other studies involving neurological disorders such as Huntington’s and Alzheimer’s Disease, CRISPR-Cas9-mediated editing was used to suppress the pathogenic expansion of the HTT gene or correct a pathogenic allele of the presenilin 1 gene (PSEN1), respectively.6,7 Other research areas such as immunology, cardiology, and hematology have all benefited from the application of CRISPR in preclinical models.

Cumulatively, the experimental evidence from preclinical studies suggests that successful genome editing can be accomplished using the CRISPR-Cas9 system, which has in turn facilitated translation and clinical evaluation of this technology.8 As a result, clinical trials utilising CRISPR technology has exploded with both ex vivo and in vivo gene editing approaches being explored in multiple therapeutic areas.9 Complex Biology and Regulatory Systems Favor Safe Plays Historical guiding principles in drug development have focused 20 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

on the underlying pathogenic mechanisms of disease and how to target the biological process to bring forth a favourable clinical outcome. One significant limitation to this approach is that the etiology of a disease can arise from multiple, diverse, and in many instances, multi-faceted underlying biological mechanisms that are impossible to co-target in today’s developmental and regulatory framework.

In melanoma, for example, the activating V600E BRAF mutation has been thought to be central in melanomagenesis.10 Tumors bearing this mutation exhibit sensitivity to downstream MAPK signalling inhibition, representing an attractive therapeutic strategy.11 However, this mutation is only present in about 50% of clinical cases.10 Likewise, in frontal temporal dementia (FTD), multiple pathogenic mutations have been described that ultimately result in the onset and subsequent progression of this debilitating disease.

Here, genetic abnormalities in C9ORF72, progranulin, and microtubule-associated protein tau are highly prevalent in FTD.12 Further, it has now been recognised that FTD is related to and shares clinical, pathological, and genetic features with amyotrophic lateral sclerosis (ALS) as 17 common genes have now been linked with susceptibility to familial forms of ALS and FTD.12 Thus, a percentage of patients with an ALS diagnosis may exhibit some degree of frontal lobe dysfunction and likewise, a percentage of FTD patients will exhibit motor neuron symptoms associated with ALS.12 These represent situations where the traditional approach of linking a single biological mechanism to a single disease state is not feasible, which makes development of therapeutics for these indications more complex from a development, and regulatory standpoint.

An even more dire situation exists for rare genetic diseases, where the development cost of a therapeutic is not supported by a favourable business case based on the number of eligible patients. In the case of Dedicator of Cytokinesis 8 (DOCK8) deficiency, which is a combined immunodeficiency, only 230 cases have been described to date.13 DOCK8 manifests as recurrent infections, autoimmunity, and malignancy through the loss of function of the protein that arise from pathogenic mutations in the DOCK8 gene.13 While regulatory approval paths for therapeutics to treat rare diseases have been relaxed to incentivise drug developers to address these unmet medical needs, significant investment is still required to develop convincing preclinical data and CMC packages worthy of regulatory review and subsequent approval for clinical evaluation.

Taken together, the biological complexity of underlying pathogenic mechanisms that drive disease progression, combined with the sheer cost and economics of drug development, creates an unforgiving and somewhat skewed landscape for the development of novel therapeutics. The Winter 2023 Volume 6 Issue 4

Research / Innovation / Development current regulatory and economic structure compels drug developers to select prevalent underlying disease mechanisms in high-impact indications with sufficient case numbers to justify the massive investment required to bring a new drug to market. Thus, substantial therapeutic voids are created, especially prevalent in rare disease syndromes or uncommon genetic mutations in more common disease types, leaving these patients with limited or even no therapeutic options.

CRISPR-Based Therapeutics Represent a Plug-and-Play Opportunity for Rapid and Transferrable Development CRISPR-based approaches have the potential to change this current regulatory and economic mantra and shift the therapeutic focus from targeting a single underlying mechanism of a disease to identifying the patient-specific genetic abnormality. The CRISPR system itself is a platformready technology in that delivery agents and endonuclease can remain constant while only the sgRNA changes based on the sequence to target. Thus, for patients with an FTD/ALS diagnosis, the underlying pathogenic genetic mutation(s) identified would dictate the sequence(s) of the variable sgRNA of the CRISPR-Cas9 cassette. Similarly, the sequence of the sgRNA in melanoma would be dependent on the presence of the activating mutation V600E BRAF or other prevalent, patient-specific upstream mutations that have been identified.

The therapeutic flexibility afforded by CRISPR-Cas9 has already started to appear in novel strategies to address complex and challenging human diseases in both preclinical models, as well as early phase clinical evaluation. In one study, for example, CRISPR-Cas9 was utilised to knockout endogenous T-cell receptor (TCR) genes and exchange patient-specific neoantigen TCRs for the personalized treatment of solid tumours.14 Similarly, preclinical studies in Alzheimer’s Disease showed that the dysregulated Aβ metabolism has the potential to be targeted by CRISPR-Cas9 through the correction of varying patient pathogenic genetic mutations that have been linked to either sporadic or familial forms of the disease.15 The potential of a CRISPR-Cas platform system, where most components are conserved and only the sgRNAs are changed to address different or multiple indications, is immense and unprecedented. However, there are still hurdles to address to realise this potential.

Platform-Based Approaches and Industry Partnerships Can Streamline Regulatory Approval With this CRISPR-Cas9-fueled revolution of highly personalised therapies showing such promise, the regulatory framework governing advanced therapeutics must evolve and change to ensure these novel ideas and technologies have the best chance at clinical translation. Center for Biologics Evaluation and Research (CBER) Director Dr. Peter Marks shares the concern that innovation in cell and gene therapy is outpacing regulatory process, creating a bottleneck for patients who are in desperate need.16 As an indication that such an evolution is possible, one need only look to the COVID-19 pandemic and the non-traditional mechanisms used to support development and eventual regulatory approval of diagnostics and vaccines. www.international-biopharma.com

Operation Warp Speed (OWS) was a three-way partnership between the U.S. Department of Health and Human Services (HHS), industry, and the Department of Defense (DOD) that facilitated vaccine, diagnostics, and therapeutics in response to the COVID-19 pandemic.17 The cornerstone of OWS was using primarily nonclinical data to move investigational solutions forward through clinical trials and, eventually, product approval in extremely compressed timelines.17 Marks’ vision is to utilise the lessons learned from OWS and apply this concept to rare diseases to facilitate therapeutic translation.

Founded in October of 2021, the Bespoke Gene Therapy Consortium (BGTC), like OWS, is multigroup partnership between the National Institutes of Health (NIH), the U.S. Food and Drug Administration (FDA), life science companies, and nonprofit organisations. The BGTC serves to develop platforms and standards that are intended to facilitate the development and clinical evaluation of gene therapies for rare diseases.18 Even though Marks is a huge proponent of this approach, he makes his cautionary view apparent that the platform approach intended to facilitate therapeutics development is a “large task,” and advises that smaller tasks need to be accomplished to achieve the overarching goal.19 These smaller tasks are heavily focused in de-risking the clinical development and manufacturing to get novel therapeutics, like CRISPR, over the finish line.19 One potential and viable avenue to de-risking multiple aspects of the development process is through strong academic, industry, and regulatory collaborations to bring together and leverage top tier expertise in all aspects of therapeutic development. To illustrate this point, the Innovative Genomics Institute (IGI), an institute composed of California Bay Area’s leading scientific research institutions, required assistance with the production of an ultra-low endotoxin cell penetrating Cas9 endonuclease for in vivo gene editing by direct injection into the brain. The system is designed to provide gene editing in the central nervous system for the treatment of neurodegenerative diseases without the common setbacks of viral-based delivery of the CRISPR platform elements.

Given the extremely sensitive application of injecting a Cas9 directly into the central nervous system, the IGI approached Aldevron for assistance with the endonuclease manufacturing project. Aldevron, an industry leader in Cas9 expression and purification, applied their extensive expertise to this project to identify and optimise a high expression method to produce a high yielding, ultra-low endotoxin Cas9 protein. The time lapse, from initial discussion with the IGI until Aldevron delivered the final product to IGI for evaluation, was less than 30 days. This collaboration led to the landmark publication of Stahl et al.20 During the study, the authors noted that the production of an ultra-low endotoxin Cas9 protein manufactured by Aldevron prevented microglial cell response and reduced humoral immunity at 21 days post injection.20 Collectively, the transient Cas9 RNPs demonstrated equivalent editing of neurons when compared to Cas9 delivered by AAV-9 and thus injection-based delivery of minimally immunogenic genome editing is a viable alternative to virus-mediated genome editing.20 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 21

Research / Innovation / Development CASE STUDY: ENABLING NOVEL CRISPR-CAS9 DELIVERY THE CHALLENGE In July 2022, Dr. Jennifer Doudna, Dr. Elizabeth Stahl, and the team at Innovative Genomics Institute (IGI) approached Aldevron for support of a collaborative research program entitled, “Correction of Neurological Disease via Allele Specific Excision of Pathogenic Repeats.” In this effort, an interdisciplinary team of biomedical scientists and clinicians from UC-Berkeley’s IGI, UCSF, and the Ohio State University endeavoured to use CRISPR-based genome editing to advance therapeutic strategies to address two devastating diseases: Huntington’s disease and amyotrophic lateral sclerosis.

Once the construct was designed and synthesized, Aldevron applied our expertise in Cas9 technology. We identified a high expression method and optimised our purification method for this construct. Knowing the goal of ultra-low endotoxin, the team included in-process endotoxin testing on fractions after first and second-step chromatography. In-process testing is a gold standard with FDA’s process analytical technology (PAT) initiative. This allowed us to identify the best quality product to move forward.

In vivo editing of somatic cells promises to be the next wave of therapies for many genetic diseases. However, the delivery of these therapies remains a significant challenge.

Recombinant adeno-associated virus (AAV) serotype 9 has greatly succeeded in gene therapy. Yet, many drawbacks remain, including costly customisation and manufacturing, immunogenicity, and limited cargo capacity. Additionally, crossing the blood-brain barrier with gene editing modalities remains an issue.

The Results Over the following year, the IGI-led research team conducted experiments on mice. They recently published results in Molecular Therapy, where they concluded:

The IGI-led team is developing a non-viral delivery system that can effectively edit neurons with minimal immunogenicity to treat diseases such as HD and ALS. The Doudna laboratory already had a cell-penetrating Cas9 variant that showed promise. However, evidence of dose-limiting immune response due to high endotoxin levels from their in-house Cas9 endonuclease production led the group on a journey to find an improved manufacturing process for their delivery system.

The IGI-led team needed a custom, highly pure, ultra-low endotoxin nuclease. And they needed it quickly.

From initial discussion to delivery of final research-grade product was < 30 days.*

These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low endotoxin Cas9 protein manufactured at scale further improve innate immunity. We conclude that injection-based delivery of minimally immunogenetic CRSPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.20 Figure S10A in the paper describes the large reduction of endotoxin in the Cas9 endonuclease using the Aldevronproduced protein versus the lab standard. It displayed 0.035 EU/mg as compared to 0.2 EU/mg, enabling dosage below the 0.2 EU/kg/hr FDA threshold for intrathecal delivery.21 This study showed the translational potential of an ultra-low endotoxin RNP as a cost-effective method.

The Action The IGI team needed a tag-less custom Cas9 variant for its cell penetrating functionality. The Aldevron stable of “off-the-shelf” products were not an option. Relying on 10+ years of experience in custom protein manufacturing, the Aldevron team got to work.

Aldevron is looking forward to continued support of this research, including producing cGMP grade material for later phases of this study.

Concluding Remarks CRISPR-Cas9-mediated gene editing has the potential to personalise therapeutics like never seen before. Through simply modifying the sgRNA sequences in the system and keeping the enzyme and delivery method the same, platform CRISPR-Cas9 can expand the reach of druggable targets to levels not seen in modern science and medicine. Thus, the genomic platformbased therapeutic approach has the potential to fundamentally change the status quo approach from treating the disease to treating the patient.

such as Aldevron, can play in instrumental role both in manufacturing and in regulatory support for Cas9 enzymes and RNP complexes.

The realisation of this dream, however, is a significant task.

Close partnerships and collaboration are absolutely required between academia, industry, and regulators to coalesce the expertise needed to ensure translational success of this as well as other nascent technologies. An established CDMO, 22 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

* Timelines vary. This is an atypical result. Contact Aldevron to discuss a more accurate timeline for your project.

With a recently announced partnership with Integrated DNA Technologies (IDT), Aldevron can now support gRNA ordering to be complexed with either stocked nucleases or custommanufactured enzymes. To deliver a therapeutic CRISPR RNP** Aldevron uses a variety of in-house methods to quantify the amount of complexed RNP, free-Cas9 and free-gRNA, during cGMP manufacturing. In addition, our proprietary release panels are compliant with 21CFR210-211 and designed to meet current draft guidance from the FDA regarding gene editing, which provides consistency of the final product. ** Aldevron provides RNPs only to customers who are duly licensed, including to make and have made RNPs, for their intended use.

Winter 2023 Volume 6 Issue 4

Research / Innovation / Development

REFERENCES 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

Jinek, M. et al. A programmable dual RNA-guided DNA endonuclease in adaptive bacterial immunity One-Sentence Summary. Science (1979) 337, 816–821 (2012).

Kantor, A., McClements, M. E. & Maclaren, R. E. Crispr-cas9 dna base-editing and prime-editing. International Journal of Molecular Sciences vol. 21 1–22 Preprint at https://doi.org/10.3390/ ijms21176240 (2020).

Weiss, T. et al. Epigenetic features drastically impact CRISPR–Cas9 efficacy in plants. Plant Physiol 190, 1153–1164 (2022).

Liu, T. et al. Targeting ABCB1 (MDR1) in multi-drug resistant osteosarcoma cells using the CRISPR-Cas9 system to reverse drug resistance. Oncotarget vol. 7 www.impactjournals.com/oncotarget/ (2016).

Feng, Y. et al. Targeting Cdk11 in osteosarcoma cells using the CRISPR-cas9 system. Journal of Orthopaedic Research 33, 199–207 (2015).

Shin, J. W. et al. Permanent inactivation of Huntington’s disease mutation by personalized allele-specific CRISPR/Cas9. Hum Mol Genet 25, 4566–4576 (2016).

György, B. et al. CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer’s Disease. Mol Ther Nucleic Acids 11, 429–440 (2018).

Sharma, G., Sharma, A. R., Bhattacharya, M., Lee, S. S. & Chakraborty, C. CRISPR-Cas9: A Preclinical and Clinical Perspective for the Treatment of Human Diseases. Molecular Therapy vol. 29 571–586 Preprint at https://doi.org/10.1016/j.ymthe.2020.09.028 (2021).

Khoshandam, M., Soltaninejad, H., Mousazadeh, M., Hamidieh, A. A. & Hosseinkhani, S. Clinical applications of the CRISPR/Cas9 genomeediting system: Delivery options and challenges in precision medicine. Genes and Diseases Preprint at https://doi.org/10.1016/j. gendis.2023.02.027 (2023).

Ascierto, P. A. et al. The role of BRAF V600 mutation in melanoma. http://www.translational-medicine.com/content/10/1/85 (2012).

Abi-Habib, R. J. et al. BRAF status and mitogen-activated protein/ extracellular signal regulated kinase 1/2 activity indicate sensitivity of melanoma cells to anthrax lethal toxin. Mol Cancer Ther 4, 1303–1310 (2005).

Gelon, P. A., Dutchak, P. A. & Sephton, C. F. Synaptic dysfunction in ALS and FTD: anatomical and molecular changes provide insights into mechanisms of disease. Frontiers in Molecular Neuroscience vol. 15 Preprint at https://doi.org/10.3389/fnmol.2022.1000183 (2022).

Biggs, C. M., Keles, S. & Chatila, T. A. DOCK8 deficiency: Insights into pathophysiology, clinical features and management. Clinical Immunology vol. 181 75–82 Preprint at https://doi.org/10.1016/j. clim.2017.06.003 (2017).

www.international-biopharma.com

14. Foy, S. P. et al. Non-viral precision T cell receptor replacement for personalized cell therapy. Nature 615, 687–696 (2023).

15. Bhardwaj, S. et al. CRISPR/Cas9 gene editing: New hope for Alzheimer’s disease therapeutics. J Adv Res 40, 207–221 (2022).

16. Jill Wechsler. FDA Eyes New Strategies to Spur Gene Therapy Development. Applied Clinical Trials https://www. appliedclinicaltrialsonline.com/view/fda-eyes-new-strategies-tospur-gene-therapy-development (2023).

17. OPERATION WARP SPEED Accelerated COVID-19 Vaccine Development Status and Efforts to Address Manufacturing Challenges Report to Congressional Addressees United States Government Accountability Office. (2021).

18. Bespoke Gene Therapy Consortium. https://www.nih.gov/ research-training/accelerating-medicines-partnership-amp/ bespoke-gene-therapy-consortium.

19. Friends of Cancer Research. AgencyIQ – CBER’s Peter Marks on advancing gene therapy, using AI, pushing accelerated approval and bespoke platforms. https://friendsofcancerresearch.org/news/ agencyiq-cbers-peter-marks-on-advancing-gene-therapy-using-aipushing-accelerated-approval-and-bespoke-platforms/.

20. Stahl, E. C. et al. Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs. Molecular Therapy 31, 2422–2438 (2023).

21. Zink McCullough, K. Calculating Endotoxin Limits for Drug Products (American Pharmaceutical Review). http://scholar.google.com/ scholar?hl=en&q=Zink+McCullough%2C+K.+Calculating+ Endotoxin+Limits+for+Drug+Products+%28American+ Pharmaceutical+Review%29

Jeff Briganti Jeff Briganti is Sr. Director, Global Strategic Marketing for Aldevron, LLC. He is responsible for developing and executing strategies to promote Aldevron’s market-leading custom development and manufacturing of plasmid DNA, RNA, and protein, supporting cell and gene therapies. Briganti joined Aldevron in 2021, bringing nearly 30 years of experience.

He spent the previous 10 years at Thermo Fisher Scientific, in various roles in Molecular Biology and Molecular Spectroscopy, most recently as Director of Strategy and Business Development, Molecular Biology. He has an MBA and a BS in Genetics, both from the University of WisconsinMadison.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 23

Research / Innovation / Development

PEER REVIEWED

Tackling the Complexities of Bringing Oncolytic Viruses to Market Oncolytic viruses (OVs) have emerged as a remarkable non-to-low-toxic and non-invasive alternative to traditional cancer treatments. These relatively new cancer therapeutics are being increasingly explored by the pharmaceutical industry to benefit from their selectivity and potential to enhance existing medicines. Despite some early clinical trial successes, numerous obstacles impede the progress of this exciting therapeutic area. In this article, Kai Lipinski, Chief Scientific Officer at ReciBioPharm, delves into the challenges that OV developers face in pursuing project success and assesses potential resolutions to bring pioneering new treatments one step closer to launch.

A Need for New Cancer Treatments Globally, cancer is estimated to cause 10 million deaths a year and accounts for every one in six fatalities.1 While traditional cancer treatments have improved survival rates, their efficacy and toxicity limitations amplify the need for alternatives or complementing treatment modalities. These include chemotherapy’s potentially disabling side effects, radiotherapy’s harm to surrounding healthy tissues and the invasive, often painful nature of surgery, underscoring the demand for innovation.

Promising approaches like chimeric antigen receptor T cell (CAR-T) and immune checkpoint blockade (ICB) therapies have limitations due to the immunosuppressive tumour microenvironment and cancer heterogeneity. Although proof-of-concept of CAR-T therapy for blood-related cancers is the reality today (CD19 target: Kymriah, Yescarta, Tecartus, Breyanzi; BCMA target: Abecma, Carvykti), successfully treating solid cancers is still to come. The potential to selectively target, infect and eliminate cancer cells while activating an immune response makes OVs a revolutionary prospect in the world of oncology. The roots of this therapy trace back to successful clinical trials as early as the 1950s.2 With an evolving understanding of molecular interactions, OVs are considered ideal candidates for combination therapies, especially when supporting traditional cancer treatments like immunotherapies. This combined approach allows for the targeting of a wider spectrum of tumours while enhancing therapeutic efficacy.3 The biopharmaceutical industry is enthusiastic about the transformative potential of OVs and is actively involved in developing new OV technologies to improve treatment outcomes. The burgeoning global OV market is projected to see a compound annual growth rate of 26.2% from 2021 to 2028, ultimately reaching $609.7 billion4. Despite a development pipeline containing over 100 OVs, their potential is yet to fully materialise into widespread commercial success. Currently, only one OV, T-VEC (Imlygic®), is approved for use in the US and 24 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

there is one conditional approval in Japan (Delytact®; Daiichi Sankyo Company, Limited) for a regenerative medical product for treating malignant glioma – interestingly, both viruses are based on HSV-1.

Tackling OV Development Challenges The push for more OVs to enter the market comes from various stakeholders. Patients with diverse cancer types would benefit directly from a broader range of potential oncology treatments while healthcare professionals would gain access to a more versatile set of treatment options. Developers, along with their investors, are eager to see OVs achieve success in the market.

However, navigating the path from preclinical development to commercialisation is challenging. OV developers embarking on this mission currently face a multitude of challenges despite the collective determination to make these promising therapies accessible to those in need.

1. Process Optimisation and Scale-up Meeting expedited clinical readiness deadlines demands rapid progress, and scaling up without losing product quality is essential to meet increasing clinical supply requirements and for further production expansion.

To address these challenges, a multifaceted approach can be beneficial: •

• •

Expertise in process optimisation, harnessed through subject matter experts (SMEs) with extensive OV manufacturing experience, can enable the efficient transfer and modification of processes at smaller scales.

Having a comprehensive understanding of the intricacies involved enables efficient problem-solving and adaptation at various stages of manufacturing, resulting in a robust process.

Effective project and program management ensures that overall timelines are met, prioritisation is maintained and escalation processes are streamlined.

Collaborative management of material supply issues, including partnering with suppliers to address potential material shortages, guarantees a smooth production flow.

2. Assay Development and Qualification The development and qualification of assays are pivotal steps in the journey to clinical readiness for OVs. Finalising assay transfer, qualification, verification and validation promptly is crucial to ensure reliable product quality assessment. The challenge of sourcing and qualifying additional outsourced testing labs for certain assays not available in-house adds complexity to the process.

Contract development and manufacturing organisations (CDMOs) represent a key resource for knowledge, providing access to specialised expertise in OV development and Winter 2023 Volume 6 Issue 4

Research / Innovation / Development manufacturing. Leveraging SME input from both the sponsor and CDMO can prove invaluable. Their expertise in assay development and qualification streamlines the process, ensuring it aligns with the project’s timelines. Establishing a strong partnership with the CDMO and maintaining reliable reaction times from both parties enhances the collaboration and enables efficient assay-related problem-solving.

An in-depth understanding of assays applicable to live viruses and their challenges is critical as each OV product comprises specific properties. On the other hand, many assays can be applied to a platform, and the available set-up from an experienced CDMO enables a plug-and-play strategy for pipeline projects.

3. Product Yield and Dosage Requirements Product yield and meeting dosage requirements are critical when developing OVs and any other product for clinical use. Designing processes to consistently produce high yields, with a high titer, low total particle: infectious particle ratio, high process recovery and a compliant process residual profile are paramount to ensuring a reliable and high-quality product, and subsequently, robust clinical supply. Moreover, modifying scaled-up processes to enable high-drug product titers is necessary to meet the dosage demands of clinical trials, particularly when targeting the intravenous administration route.

To tackle these challenges effectively, a streamlined process development approach is imperative. Conducting multiple process development studies optimises both upstream and downstream parameters, ensuring the development of a robust process capable of meeting product yield and quality requirements. High flexibility, from both the CDMO and sponsor, is key in swiftly implementing necessary process adaptations to meet dosage requirements and aligning the production process with the clinical trial needs.

This comprehensive approach ensures that OV developers can successfully address these challenges and advance their products from the research and development to clinical phases, offering promising new treatments to patients in need.

Having access to extensive process data for specific OV types allows a platform approach to be extended to future clients with minimal development activities before commencing GMP manufacture.

4. The Role of Collaboration in OV Development Overcoming the hurdles on an OV’s journey to clinical phases – including expedited process development, limited access to expertise, scaling production, optimising product yield and addressing complex technical intricacies – demands specialised solutions. These challenges underscore the multifaceted nature of OV development, requiring tailored strategies and expertise to surmount.

The success of innovative OV therapies heavily relies on a joint effort, not just between researchers and developers but also across different entities involved in the process, such as preclinical development (PD), quality control (QC), production, project management and quality assurance/qualified person (QA/QP). www.international-biopharma.com

•

•

•

Mutual dependence for progress: A symbiotic relationship between shareholders with shared responsibilities forms the cornerstone for a united approach in tackling the multifaceted hurdles of OV development.

Transparent and constructive exchange: Open and transparent communication channels from all stakeholders foster an environment of constructive problem-solving.

This allows for the swift identification of challenges and enables a proactive approach towards finding a resolution.

Specialised, expert and diverse collaborators: Active collaboration between SMEs from various backgrounds is essential in deciphering and resolving the technical intricacies that come with OV development, driving innovative solutions in this complex area.

Ultimately, the success of OV development relies not just on scientific breakthroughs but on a unified commitment to addressing challenges. This involves collaborative efforts driven by a shared goal of bringing potentially life-changing treatments to fruition.

The Measurable Impact of Overcoming OV Development Challenges Overcoming the hurdles that can arise in process optimisation, assay development and qualification, and ensuring product yield, has significantly advanced the OV field. This has led to meaningful progress and benefits for both research and potential patient outcomes.

One of the most significant impacts lies in the initiation of clinical trials. Overcoming developmental hurdles ensures timely access for patients to potentially groundbreaking therapies, enhancing the potential for positive health outcomes.

These advancements in clinical trial readiness stem from the more efficient and quality-assured manufacturing process. This enhancement significantly reduces the risk of batch failures, ensuring consistent product quality and meeting rigorous regulatory standards.

Furthermore, resolving scalability challenges has secured a sustainable and scalable supply chain for OVs, accommodating both current clinical dosing requirements and potential dose escalation for future medical needs. These improvements in manufacturing processes have directly contributed to ensuring a reliable and consistent supply of OVs for advancing clinical needs. In particular, shifting from adherent to suspension cell line conditions has catapulted the scalability and robustness of OV production. Such cell line options comprise currently mostly HEK293, HeLa and A549, and proprietary cell lines like CAP®, EB66® and AGE1.CR®.

Moreover, optimising process efficiency and enhancing assay development has significantly increased product yield, meeting stringent dosage requirements and fostering the qualification of advanced assays. These improvements highlight a key aspect of OV development, ensuring not only higher product yields but also the necessary tools to gauge their efficacy accurately.

Additionally, collaborative efforts have played a pivotal role, encouraging synergy between stakeholders, researchers and developers, contributing to overcoming key obstacles and achieving milestones in the OV development journey.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 25

Research / Innovation / Development

Collaboration has been instrumental in navigating challenges, emphasising the importance of collective efforts in driving OV development forward.

The combined impact of overcoming these challenges marks a transformative stride in OV development, showcasing advancements across multiple fronts and paving the way for more promising OV therapies.

The Future Outlook The increase of OV therapies in the pipeline has the potential to help many people living with cancer. However, traversing the pathway to clinical approval requires developers to understand the obstacles and decide how they can be overcome.

The numerous challenges facing OV developers encompass a spectrum of complexities, ranging from expediting process development to limited access to those with the required expertise, and beyond. These obstacles can appear formidable, but they are not insurmountable. Those equipped with the right blend of efficient project management, collaborative partnerships, expertise and flexibility, can successfully address these barriers.

The proficiency and expertise held by dedicated professionals and institutions are instrumental in charting a path through the hurdles, offering novel insights and solutions to intricate problems. Flexibility, outstanding willingness and a high level of project/product identification serve as cornerstones in adapting to dynamic situations and addressing evolving requirements, ensuring adaptability in the face of unforeseen challenges.

26 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Through these concerted and combined efforts, OVs are poised to offer promising new treatments to cancer patients.

Further development in this area holds the potential to usher in a brighter future for oncology.

REFERENCES 1.

2.

3.

4.

https://www.who.int/news-room/fact-sheets/detail/cancer Kelly E, Russell SJ. History of oncolytic viruses: genesis to genetic engineering. Mol Ther. 2007; 15(4): 651-659.

Zhang B, Cheng P. Improving antitumor efficacy via combinatorial regimens of oncolytic virotherapy. Mol Cancer. 2020; 19, 158. https://www.biospace.com/article/oncolytic-virus-therapiesmarket-growth-at-a-cagr-of-26-2-percent-during-forecastperiod-2020-2028/

Kai Lipinski Kai Lipinski has a wealth of experience in viral vector manufacturing from a variety of roles before he joined ReciBioPharm. He served as Principal Scientist at Cobra Biologics, focusing on upstream process development for virus and mammalian protein expression projects. Prior to that, Kai worked as Senior & Principal Scientist at ML Laboratories, where he was responsible for the development of targeted adenoviral vectors for cancer gene therapy approaches. At Vibalogics, Kai is central to the establishment of virus Process Development and Manufacturing capabilities, technical developments and the acquisition of many key clients. Kai has a PhD in Transcriptional Regulation by Adenoviral E1A Proteins, and a Post-Doc, also on Transcriptional Regulation, from the University of Duisburg-Essen.

Winter 2023 Volume 6 Issue 4

A RE Y O U LO O K I N G F O R E X P E RT S I N

MICROBIAL PRODUCTION?

CONTRACT DEVELOPMENT AND MANUFACTURING OF BIOPHARMACEUTICALS Richter-Helm is a Germany-based GMP manufacturer specialized in products derived from bacteria and yeasts, with a proven 30-year track record.

Count on us to flexibly provide a comprehensive range of services and customized solutions.

Clients worldwide have already benefited from our commitment to good manufacturing practice and total transparency. Our work focuses on recombinant proteins, plasmid DNA, antibody fragments, and vaccines.

Richter-Helm consistently works to the highest standards of pharmaceutical quality.

Contact us +49 40 55290-801 www.richter-helm.eu

LEARN MORE ABOUT OUR SERVICES AND www.international-biopharma.com CAPABILITIES

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 27

Application Note

Primary Packaging Solutions for Intravitreal Administration of Ophthalmic Drugs – Critical Features of Prefillable Syringes One of the probably most sensational organs of the human body are the eyes. Visual perception, reception and processing of optical stimuli from our environment takes place via the eyes. Therefore, it is even worse for affected persons when this is negatively influenced by diseases or degeneration. Due to the increasingly aging society and the increased prosperity worldwide, the number of chronic and age-related diseases in the field of eye diseases is also rising.

According to WHO, at least 1 billion people suffer from eye disease. One of the most prominent retinal disease is macular degeneration, in which the central visual acuity of an eye is partially or completely lost. Another 500,000 - 600,000 patients with diabetes mellitus suffer from eye damage as a secondary disease, the so-called diabetic retinopathy. The advent of novel drug technologies such as gene therapies or RNAi-based approaches are opening treatment options in the field of genetic diseases (e.g. retinitis pigmentosa), among others.

Prefilled Syringes (PFS) Used in Ophthalmology PFS used in the area of ophthalmology are primarily used for cataract surgeries in the anterior eye area or with vascular endothelial growth inhibitors to treat inter alia wet macular degeneration intravitreally. In the case of cataract surgeries, the injection of viscous hyaluronic acid prevents the collapse of the anterior chamber and simplifies the insertion of artificial intraocular lenses. For macular degeneration, monoclonal antibodies (mAbs) or recombinant fusion proteins are injected

into the vitreous body to inhibit the adverse growth of blood vessels and in this way maintain the sight of the patient. The hyaluronic acids used for cataract operations are highly viscous but are not especially sensitive to potential chemical interactions.

Conversely, the monoclonal antibodies (mAbs) and recombinant fusion proteins used for the therapy are biotechnologically manufactured and often sensitive to drug-container interactions over shelf-life. Both therapies are very different, although the eye is treated in both cases. Prefilled syringes have proven themselves over the last years very well as a primary packaging solution, making drug delivery in the area of ophthalmology more user-friendly and decreasing risks in regard to dosing and administration. In ophthalmology they are injected with fine needles repeatedly at intervals of several weeks directly into the interior of the eye (Figure 1). In these cases, low dosage volume requires highly precise syringe dimensions and scaling. Both treatment areas are subject to strict requirements regarding the allowed particle load.

Our whitepaper “prefillable syringes – critical features for ophthalmological applications” provides an overview of requirements of PFS used in this therapeutic area as well as highlighting the benefits associated by using PFS instead of the conventional way of vials and Polypropylene syringes.

Lowest particle levels to ensure compliance with USP <789> and also reduce risk for the drug product and patients is one of the key benefits for PFS with baked-on siliconisation (BOS) and silicone-free syringes in Glass and COP compared to polypropylene syringes commonly used for repacking.

Figure 1: Major applications of ophthalmological drugs – injection into the anterior section of the eye during cataract surgery and injection of Anti-VEGF proteins into the vitreous body 28 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Winter 2023 Volume 6 Issue 4

Application Note syringes were evaluated and compared to baked-on siliconized glass syringes. The subvisible particles were measured according to USP <789> after 3 months of real time storage using the light obscuration method, see graphics 2, 3 and 4.

The particle count for silicone-free syringes is exceptionally low. BOS syringes have shown particles loads comparable to the silicone-free options, suggesting that both are well suited for ophthalmological applications. Both comply well with USP <789> requirements and can certainly be considered as an alternative for siliconised PFS for ophthalmological injections already existing on the market.

Figure 2: Number of subvisible particles ≥10µm/1ml. USP <789> requirements for drug/device combination products: max 50µm/ml

Figure 3: Number of subvisible particles ≥25µm/1ml. USP <789> requirements for drug/device combination products: max 5µm/ml

Dr. Christian Redeker

Figure 4: Number of subvisible particles ≥50µm/1ml. USP <789> requirements for drug/device combination products: max 2µm/ml

Silicone Free Syringe Systems Although silicone oil is harmless to the human body, the repeated nature of intravitreal injections for the treatment of chronic ophthalmic diseases and the fact that the vitreous body is a small, confined space can raise the risk of such therapy complications as floating particles, endophthalmitis and high intraocular pressure. Especially to address the requirements of the next generation drug technology products such as AAV-based gene therapies and LNP-encapsulated RNAi gene regulating therapies it is even more favorable to eliminate silicone oil in all components of a syringe system by ensuring full functionality.

In a broad set of testing three different stopper options (1, 2, 3) for silicone-free configuration used in glass and COP www.international-biopharma.com

Dr. Christian Redeker has a doctorate in molecular biology in Hanover, has been with Gerresheimer for 7 years in various positions. In his current position as Global Product Manager Syringes, he optimizes Gerresheimer's glass syringe portfolio and develops strategies to be able to offer the best possible products to meet the new challenges in the glass syringe sector.

Contact: Dr. Christian Redeker, Global Product Manager Syringes Business Development T: +49 5223 164 373, M: +49 175 46 51 755 Christian.redeker@gerresheimer.com Medical Systems, Gerresheimer Bünde GmbH Erich-Martens-Str. 26–32, 32257 Bünde www.gerresheimer.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 29

Therapeutics

PEER REVIEWED

A Novel Approach to Inhalation Therapy

The adaptation of e-cigarettes as drug delivery devices holds great potential for inhalation therapy. So, can we apply the advances made in nicotine delivery to other active substances?

Inhalation therapy is used to treat patients with both acute and chronic respiratory diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, and more.

Respiratory disease is a huge global issue – an estimated 328 million people suffer from COPD globally, with many cases undiagnosed.1 WHO reports COPD as the third leading cause of death worldwide; 3.23 million in 2019, which was 6% of total deaths globally. According to WHO, asthma affected an estimated 262 million people globally in 2019 and caused 455,500 deaths.

Both of these conditions are primarily treated with inhalation therapy. For the treatment of asthma, patients typically inhale bronchodilators and antiinfl ammatory agents. Whereas with COPD, treatment generally is via the inhalation of bronchodilators, steroids, and antibiotics.

Overall, inhalation therapy brings several benefits over tablets and capsules. There is the obvious advantage of target organ delivery for afflictions of the throat, lung, and oesophagus.

However, inhalation therapy can also have distinct advantages for other conditions, although not delivered directly to the target organ. The delivery of therapies into the alveolar spaces of the lung results in rapid absorption to oxygenated blood.

This allows the active substance to reach the target organ quickly and relatively intact. In comparison, intravenous injections rely on the delivery of the substance into deoxygenated blood, which first requires passage around the body: through the heart, lungs, and then to the target organ. This relative delay can result in significant metabolism of the active substance before it reaches the target organ. Through inhalation, efficacy can be achieved with a lower dose, potentially reducing the risk of side effects and adverse events.

The deposition of the active substance is essential in achieving efficacy. The correct administration of an inhaled drug to the patient depends on several factors related to the drug’s formulation, device design, and patient use. For the treatment of lung disease, aerosol droplets must be of a size where they are not deposited on the back of the throat or in the bifurcations of the bronchi. This ensures that as much of the active substance is delivered to the alveoli as possible. In contrast, to treat a disease related to the throat or oesophagus, the particle size should be larger, to ensure deposition in the upper airway limiting the dose delivered to the lung.

30 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Current Inhalation Technology Inhalation therapy is traditionally performed by four types of devices: pressurised metered dose inhalers (pMDI), soft mist inhalers (SMI), dry powder inhalers (DPI), and nebulisers.

Many different inhaler devices and medications are available to treat asthma and COPD, with over 230 drug delivery system combinations available. Despite this, disease controls in clinical practice are often unsatisfactory, where a key determining factor is the match or mismatch of an inhalation device with the needs of an individual patient.2 The choice of inhaler device is essential, and can significantly impact the effectiveness of the treatment.

Different groups of devices bring specific advantages and disadvantages. For example, nebulisers, which turn liquid medicines into mist, are easier to inhale. They are therefore commonly used in situations where delivery with a classic inhaler might be difficult, such as to administer salbutamol to patients who are struggling to breathe during an asthma attack, or to frail or older patients unable to inhale deeply.

One historic downside of nebulisers is that they are typically large, desk-based systems, and therefore less suitable for home use. There is a need for new innovations in inhalation therapy to address the disadvantages of current technologies. Naturally, it is easier to adapt the inhalation device to improve the performance of generic drugs than to develop a new drug.

There has consequently been substantial evolution in inhaled medicine dispensers, and there are numerous technical innovations in development – for example, handheld nebuliser technology.3 Researchers have suggested three main areas in which we can expect innovation: device engineering and design, chemistry and formulations, and digital technology.4 New Therapeutic Options Novel methods of inhalation therapy may help expand the number of medicines delivered by aerosol to treat a broader range of conditions. For example, sildenafil (Viagra®), was designed as an antihypertensive, but the high doses required for the drug to be efficacious when administering it systemically via tablet caused such notable side effects that the drug is instead prescribed to treat erectile dysfunction. Researchers are investigating the possibility of sildenafil delivery to treat hypertension using inhalation therapy at a lower dose than is given orally.5 Take corticosteroids as another example. Inhaled beclomethasone is routinely prescribed as a mainstream treatment for asthma and COPD, with very little to no side effects. For patients suffering exacerbations, the most common course of action is to prescribe the closely-related prednisolone as tablets. These are typically administered for around four weeks.

Winter 2023 Volume 6 Issue 4

Therapeutics

However, the side effects can be severe and include risk of infection, increased appetite, higher blood pressure, and mood swings. In addition, therapy must be slowly phased out to avoid side effects or withdrawal symptoms at the end of the treatment.

This example clearly shows the potential of lower-dose inhalation to reduce systemic adverse events.

Learning from Nicotine So where can pharmaceutical companies look for inspiration when developing novel inhalation therapies? One, perhaps surprising, answer, is to the nicotine industry. One of the reasons smoking is so addictive is that it is one of the most effective and efficient methods of delivering a drug to the lung.

Recent research from the industry has identified that one of the reasons smoking provides such efficient and quick drug delivery is the way that the nicotine coalesces with water molecules. This inspired research into whether the pharma industry could develop an aerosol where the drug binds to water in a similar way, to improve the efficiency of drug delivery of other active substances.

Boehringer Ingelheim have previously developed the soft-mist inhaler, and bronchodilator tiotropium bromide. This combination was a huge breakthrough for the treatment of asthma and COPD, and has been a major success. It started a revolution in inhalation therapy: the first time an active substance was delivered through inhaled water droplets. The question now is, how can we emulate this with other drugs?

We’ve already established that cigarettes are a hugely effective drug delivery mechanism, in the case of nicotine.

Interestingly, e-cigarettes are not far behind. This poses an interesting question: instead of nicotine being delivered through e-cigarettes, could we administer other active molecules?

Using similar technology would be a cheap, cost-effective, and www.international-biopharma.com

potentially very efficacious way of getting active substances to the lung. It may also be easier for patients to administer than current inhaler technology. For example, inhaler use depends on very close coordination between the trigger and inhalation, and a deep breath is required.

While there is a prescription route available for e-cigarettes via the MAA in the UK, this is currently for using e-cigarettes during smoking cessation. However, there is no reason for pharma businesses to turn away from trying to deliver other active substances using technologies similar to that used within an e-cigarette.

Non-heated Technologies Non-heated vaping technology based on ultrasonics, piezoceramic mesh, and micro-nozzles, similar to that used in medical nebulisers, is a growing area. These technologies atomise liquid, rather than vaporising it, using mechanical action to create a fine mist or spray, thus removing the need for conventional heater coil technology. Atomisation technologies allow a more controlled dosage, and an improved ability to manipulate and control particle size to optimise where the drug will be deposited. Non-heated technologies are more repeatable and consistent, and may also reduce the risk of irritation or harmful emissions formed through thermal degradation of the liquid.

E-cigarette technology based on piezo technology might be best suited to inhalation therapy due to the ability to fine-tune the mesh, vibration frequency, or other parameters within the device, depending on the liquid formulation.

The evolution of e-cigarette technology is happening rapidly, and device consistency and DDU are unlikely to remain a concern for many years. The second step is then to control how much drug is administered overall.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 31

Therapeutics Taking The Leap There is still research to be done in drug formulation and suspension. The design and synthesis of inhaled drugs is challenging, as many factors must be considered. Such factors include the target, mode of action, length of action, and how to achieve it (pharmacokinetics vs receptor or enzyme binding kinetics, limited solubility vs soluble compounds), the desired onset of action, and synthetic risk.7 Wherever possible, the development of the device and the drug should be performed in parallel.

The nicotine industry is innovating rapidly, with the development of novel e-cigarette technology and the creation of smart devices. Previous pharmaceutical breakthroughs have been inspired by research performed in the nicotine industry.

What we need now is a major pharmaceutical player to commit to bringing this technology to market. The hardware and software already exist, and so there is the potential for breakthrough products in the next five years. This will be welcome news to the hundreds of millions of patients worldwide suffering from respiratory disease, and the clinicians and healthcare providers who treat them.

REFERENCES 1.

2.

3.

4.

5.

Improving Patient Compliance with Smart Technology As with inhalers, treatment efficiency depends on how effectively the patient adheres to the prescribed regimen. Many patients prescribed medication twice daily will not reliably take the prescribed dose. An observational study into COPD patients found that almost half of patients made at least one error, with 50% of these errors relating to the technique, and 19% related to both device and technique.6 Though the risks of improper administration of a Ventolin inhaler might be low, it is significantly increased for patients using corticosteroid inhalers.

To prevent patients from continuously puffing on a medicated e-cigarette, manufacturers can build firmware into devices that control the amount of drug administered. Incorporating Bluetooth into an e-cigarette means the user can connect their device to an app that monitors relevant data. For example, how much power has been supplied, length of inhalation, frequency of puff, and number of inhalations per day. As well as improving how drug administering is monitored and controlled, the patient and physician can gain insight from this information. Unlike with an inhaler, where ten puffs a day might be prescribed, with an e-cigarette, it may be time-based – 60 seconds per day, for example. Using smart devices as part of a therapy regimen, with either an inhaler or e-cigarette technology, can overcome the issue of patient compliance in a way that no current therapy can. Clinicians could see from device data whether the patient is forgetting their medication, or taking the drug as prescribed and it not working.

32 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

6.

7.

Quaderi SA et al, The unmet global burden of COPD, Global Health, Epidemiology and Genomics, 2018.

Cataldo D et al, How to Choose the Right Inhaler Using a PatientCentric Approach? Advances in Therapy, 2022 Cazzola M et al, Advances in pulmonary drug delivery devices for the treatment of chronic obstructive pulmonary disease, Expert Opin Drug Deliv, 2020 Biddiscombe MF et al, Is there room for further innovation in inhaled therapy for airways disease? Breathe, 2018 Rashid J et al, Inhaled sildenafi l as an alternative to oral sildenafil in the treatment of pulmonary arterial hypertension (PAH), J Control Release, 2018 Lindh A et al, Errors in inhaler use related to devices and to inhalation technique among patients with chronic obstructive pulmonary disease in primary health care, Nursing Open, 2019 Pasqua E et al, Developing inhaled drugs for respiratory diseases: a medicinal chemistry perspective. Dru Discov. Today. 2021

Paul Hardman Paul Hardman has 10 years of experience in developing inhaled pharmaceuticals, across API particle engineering, formulation development and scale-up, and device design including interaction with the formulation to enable products to target either systemic absorption or local effect in the lung Passionate about product quality and understanding a product chemistry, from product design to its intended function, how the chemistry changes over time, and assessing any special features Leading the European Committee for Standardisation working group on extractable and leachable compounds in vaping products Currently leading studies related to product chemistry across a wide range of consumer and medicinal products at Broughton.

Email: phardman@broughton-group.com

Winter 2023 Volume 6 Issue 4

MISSION

#OneStopShop

for stability and release testing Since 1995 A&M STABTEST has been a household name for highest quality analytical services “Made in Germany”. Our 340 strong team of dedicated specialists and stateof-the-art equipment get your projects done on time and in budget. We are your #OneStopShop solution for:

•

Stability and release testing of small molecule and biological drug substances and drug products

•

Analytical development and method validation

•

mRNA-LNP ready methods acc. to draft USP guideline

•

Dedicated cell-based potency assay group

•

ICH-stability storage and logistics service

•

Laboratory dedicated to inhaled drug testing (DPI, MDI, Nebulizer)

•

GMP compliant LC-MS service for extractable and leachable testing, protein characterization, identification of unknowns and quantification of impurities and excipients

•

Functional testing of container closure systems, prefilled syringes and injection devices (CCIT, force measurements)

•

Analysis of ATMPs

#CONTACT www.am-labor.de anfragen.BM@am-labor.de A&M STABTEST; Kopernikusstrasse 6; 501026 Bergheim; Germany www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 33

Manufacturing & Processing

Adapting to the Ever-evolving Cell and Gene Therapy Landscape Over the past 10 years, the cell and gene therapy (C&GT) space has grown rapidly, with the number of innovative medicines entering the development pipeline steadily rising year on year.

The potential of C&GTs continues to expand as our understanding of molecular biology and genetic engineering techniques grows and manufacturers adopt innovative technologies. As well as improving the safety, efficacy and manufacturability of these therapies while helping to broaden patient access, advancements in the C&GT space are driving trends and introducing new challenges. From meeting changing regulatory requirements to adopting strategies to minimise manufacturing costs, C&GT developers must adapt to navigate these novel difficulties and continue to provide life-changing medicines to current and future patients.

In this article, Andelyn Biosciences examine the drivers behind current trends in the C&GT space and the obstacles developers and manufacturers face on their journey to market. Leveraging its unique insight, Andelyn Bioscicences emphasizes the importance of implementing specific strategies to quickly adapt to changing C&GT needs as they emerge.

Three Decades of Rapid Expansion Following the success of the first approved gene therapy procedure in 1990 to treat a patient suffering from severe combined immunodeficiency (SCID), the biopharma industry has seen a steady increase in resources dedicated to unlocking the potential of C&GTs.1 Over the last decade, the biopharma industry has intensified its focus on next-generation technologies such as C&GTs and precision medicines. There are now 26 gene therapies and 63 non-genetically modified cell therapies approved for clinical use globally.2 These revolutionary therapies target a wide variety of indications, from rare diseases to cancers and neurological disorders.2 Prominent factors driving the demand for C&GTs include the rising prevalence of cancer worldwide and the expanding population of chronic disease patients.3 This growing demand is reflected in the projected expansion of the global C&GT market from US$13 billion in 2022 to US$62.5 billion by 2032 at a compound annual growth rate of 22.8%.4 Many of the therapeutics behind the recent growth in the C&GT market require an extremely skilled workforce, state-ofthe-art technologies and advanced consumables, including plasmids and viral vectors. As a result, meeting the expanding demand for C&GTs has increasingly relied on outsourced development, manufacturing and testing organisations offering 34 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

the necessary capacity, technical capabilities and expertise to support production.

Navigating the Challenges of a Novel Therapeutic Space Although the C&GT market has grown immensely in the last 30 years, it is still relatively new compared with other therapeutic areas. Our understanding of the treatments’ mechanisms of action and delivery has rapidly transformed in this time. As a result of this transformation, novel challenges have also surfaced in the development and manufacturing of these new medicines.

A Lack of Necessary Expertise and Experience Staffing has been a persistent challenge in the biopharma industry and it has been felt strongly in the burgeoning C&GT space. Even now, few companies possess the technologies needed to scale commercially, and even fewer have experience working with multiple vectors and different serotypes in those systems. As a result, finding staff with relevant C&GT experience is a significant challenge for developers.

To attract talent with a breadth of late-stage and commercial experience across different geographies, development and manufacturing organisations supporting C&GTs must carefully design recruitment and retention models.

A Need for Clarity from Regulatory Bodies The rapid advancement of C&GTs has forced regulatory bodies to quickly adapt guidance to meet the changing requirements of their production. Although various agencies including the FDA and EMA have created regulatory frameworks for C&GT development and manufacture, there are still some areas where clarification may be needed to avoid delays as projects progress toward the market. In part, this has been attributed to the predominant familiarity of regulators with assessing processes used to make traditional biologics like monoclonal antibodies (mAbs) as opposed to novel therapies.5 With the increasing demand for C&GTs globally, it is critical developers and manufacturers build strong relationships with the relevant regulatory bodies to ensure smooth delivery to market. Worldwide, various regulatory agencies have shown they are willing to work with innovator companies creating programs designed to improve interactions between the agency and therapeutic developers.

Adopting and Adapting Technologies to Scale Production Initially, many of the systems and tools utilised in developing, manufacturing and testing C&GTs were derived from academic settings. It quickly became apparent that many of these techniques and technologies were unsuitable for scaled production, especially when aiming to provide enough material for a global patient population.

As a result, industry leaders have focused on designing advanced instruments to enable drug developers to improve Winter 2023 Volume 6 Issue 4

Manufacturing & Processing

the manufacturability of these innovative medicines. In the past five years alone, there have been significant advances in the techniques and technologies used throughout C&GT development, from molecule screening to purification. For example, in viral vector production, suspension cell culture platforms have gradually replaced adherent processes as the favoured cell type to enable scalability.

their models to provide the flexibility needed to serve both relatively small-batch manufacturing and larger indications.

However, there is still a long way to go to fully realise the potential of these cutting-edge therapeutics. Looking to the horizon, C&GT producers must continue to demonstrate flexibility, adapting to overcome the challenges ahead. This includes:

Additionally, many of the technologies used in C&GT development and manufacturing have shifted to single-use systems to limit cross-contamination and improve safety.

Although single-use options have the potential to shorten timelines by reducing the cleaning and sterilisation burden, they have also complicated the raw material supply chain, driving extended lead times for C&GTs.

•

There’s Still Change to Come in the C&GT Space As production needs of C&GTs have risen, developers, manufacturers and supporting organisations have adjusted www.international-biopharma.com

Integrating Automation Although there are areas where automation has been widely adopted within today’s C&GT manufacturing environment, the industry still has a long road ahead to match the level seen in the production of traditional biologics like mAbs and recombinant proteins.

A key driver behind the integration of enhanced processing and automation in C&GT manufacturing is the need to provide broader patient access to potentially life-changing treatments. As viral vector technologies continue to expand INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 35

Manufacturing & Processing from targeting rare diseases to treating more prevalent indications, automation will need to be embraced to meet demand.

Additionally, the extensive costs of gene therapies can benefit from the integration of automation. With gene therapies costing between $1 million and $2 million on average per dose, automation will play a key role in driving down manufacturing costs and providing broader patient access.6 Automating the manufacturing process will reduce the need for highly specialised labour, helping to reduce costs while improving process reproducibility and predictability.

•

Further Improving Manufacturability In addition to adopting automation technologies, C&GT developers and manufacturers must implement other strategies and tactics to enhance the manufacturability of these revolutionary medicines.

One strategy is the use of producer cell lines where there has been an increasing interest. These producer cell lines are equipped with genes needed for viral vector protein expression that are stably integrated into their genomes.

In contrast to relying solely on transient transfection processes, stable producer cell lines can improve product yields and quality by reducing batch-to-batch variability and simplifying upstream culture and harvest. Over time, these advances can reduce the cost of goods (COG) significantly and, consequently, lower manufacturing costs.

However, we have only just begun to understand the potential of C&GTs. As new trends emerge, producers must consider adopting tactics to further improve therapy manufacturability. This, in turn, will broaden patient access and continue changing patients’ lives. Finding the support of development and manufacturing partners adopting these techniques will be critical in meeting the rising demand for C&GTs anticipated on the horizon.

REFERENCES 1.

2.

3.

As well as cell line improvements, the incorporation of manufacturing methods designed to improve cost-efficiency can further boost patient access in disease areas where the price of therapeutics is a limiting factor.

•

Centralisation for Robust Supply Chains A robust supply chain is critical to avoid potential delays and to ensure the reliable and secure delivery of C&GTs to key milestones and subsequently, the patients who need them.

As well as developing a robust supply chain, C&GT developers and manufacturers should focus on adopting a centralised model to minimise delays, particularly when striving to meet growing demand. A centralised manufacturing hub can reduce the time surrounding tech transfer, as well as enable transparent and swift communication between all groups involved in production.

By consolidating their analytical repertoire and bringing critical assays in-house through centralisation, developers and manufacturers can also reduce timelines and strengthen control of vital lead times.

Preparing to Embrace Tomorrow’s C&GT Challenges The C&GT space has changed rapidly following the success of the first approved gene therapy over three decades ago.

Developers, manufacturers and all organisations offering support services in the production of these innovative therapies have had to demonstrate flexibility and agility to adapt to the quickly evolving C&GT landscape.

36 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

4.

5.

6.

Anderson WF. September 14, 1990: The beginning. Hum Gene Ther.

1990 Winter;1(4):371-2. doi: 10.1089/hum.1990.1.4-371. PMID: 1981846.

Gene, Cell, and RNA therapy landscape report. Q2 2023. American Society of Gene and Cell Therapy. https://www.globenewswire.com/en/news-release/2023/ 04/11/2644248/0/en/Gene-Therapy-Market-Revenue-to-CrossUSD-49-3-Bn-Globally-by-2032-CAGR-of-25.html https://www.bloomberg.com/press-releases/2023-05-03/ cell-and-gene-therapy-market-projected-to-grow-at-a-cagr-of-228-and-reach-us-62-5-billion-by-2032 https://bioprocessintl.com/bioprocess-insider/regulations/cell-andgene-sector-needs-regulatory-clarity-says-andelyn/ https://www.genengnews.com/insights/cell-and-gene-therapymanufacturing-costs-limiting-access/#:~:text=Cell%20and%20 gene%20therapies%20are,and%20%242%20million%20 per%20dose.

Andelyn Biosciences, Inc. Andelyn Biosciences is a full-service cell and gene therapy CDMO focused on the development, characterisation and production of viral vectors for gene therapy. With more than 20 years of experience, Andelyn's deep scientific expertise has resulted in the production of cGMP material for more than 450 clinical batches and 75 global clinical trials. Operating out of three Columbus, Ohio facilities, Andelyn supports its clients in developing curative cell and gene therapies from concept through plasmid development and manufacturing, process development, and cGMP clinical and commercial manufacturing.

Andelyn's versatile capabilities include cGMP manufacturing capacity for both adherent and suspension processes up to a 2,000-liter capacity. An advanced digital model, quality system, full regulatory support and supply chain vertical integration help Andelyn accelerate the development and manufacturing of its clients' innovative cell and gene therapies. For more information, visit andelynbio.com.

Winter 2023 Volume 6 Issue 4

LISTEN, DEVISE BREAKTHROUGH

Our approach to advancing your therapies beyond milestones to transforming lives

Discover Catalyst

A biotech-focused Oncology CRO

Resourcing and functional solutions

helping to bring next-generation therapies to cancer patients in need

helping to drive healthcare innovations for patients in need

www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 37

Manufacturing & Processing

Adapting to Supply Chain Issues Surrounding Antibody-based Therapy Production The complexity and diversity of antibody-based therapies have been increasing with the growing understanding of the intricate mechanisms involved in immune responses, the discovery of novel therapy targets and the development of new technologies for antibody production. As a result, the biopharmaceutical industry has placed greater significance on advancements in manufacturing and collaborations within the supply chain. However, introducing new products or materials into a pre-existing process can be time-consuming and costly, as regulatory agencies must be notified and the entire manufacturing process reapproved.

Better access to innovative purification materials is crucial for delivering new antibody therapeutics to market, and the patients who need them. To overcome regulatory hurdles and adopt new materials into current processes with greater efficiency, the biopharmaceutical industry is exploring various strategies.

In this article, Aaron Moulin, Field Application Specialist at Purolite, discusses the need to establish robust supplier networks that will support streamlining biologics development and manufacturing processes. He also explores some of the emerging purification challenges associated with developing novel antibodies, emphasizing the importance of implementing a quality-by-design (QbD) approach when adopting new resin technologies.

Finding Support Within a Robust Supplier Partnership The ongoing challenges caused by the COVID-19 pandemic, such as labour shortages, supply chain disruptions, and increased competition have contributed to the need for manufacturers to innovate and optimise their manufacturing techniques and business practices.

A key focus for manufacturers should be ensuring supply chain resilience for bioprocess resins by sourcing multiple suppliers of key raw materials, securing domestic manufacturing capabilities, and manufacturing on multiple continents if necessary.

Downstream processing (DSP) techniques such as chromatography are used in monoclonal antibody (mAb) production to remove unwanted contaminants and to obtain a highly purified end product during purification. Manufacturers should aim to intensify their purification process by adopting processing methods that achieve sufficient speed, high throughput, and efficient use of facility space. As DSP plays a critical role in maintaining product safety, future-proofing is essential; this relies on robust supply chain networks.

It is crucial to choose products and suppliers with a proven track record in innovation to meet the ongoing demand for chromatography resins with high binding capacities and mitigate 38 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

manufacturing bottlenecks in downstream purification. This way, manufacturers can ensure a bright future for mAb bioprocessing in the face of ongoing global uncertainty.

The Emergence of Novel Purification Methods Novel antibody-based therapies are increasing in prevalence, reflected in the bispecific antibody market for cancer therapies, which is predicted to grow by over $400 million by 2027.1 With the complexity of antibody-based therapies steadily increasing, classic purification methods may no longer be viable.

Chromatography resins made of cross-linked agarose, glass, or polymer beads bound to a Protein A ligand via a linker are commonly used in mAb purification. Protein A exhibits a strong affinity for antibody-based materials and can form a complex at pH levels ranging from 5 to 8, with the bound antibody being eluted when the pH is lowered to acidic conditions. This method is generally employed in the initial stages of development and maintained throughout commercial production.

Novel antibody-based therapies do not always share the same characteristics as mAbs. Therefore, innovative approaches to chromatography, such as alternative Protein A resins that allow for elution at higher pH levels, may be required to maintain high yields.

High pH technology resins also offer increased capacity for binding antibody-based products, allowing for increased productivity and reduced resin usage. This can result in cost savings, as less resin is required to achieve the desired yield of purified product.

However, sourcing and introducing new resins into preapproved processes can be difficult as these technologies are not easily available and may not have off-the-shelf solutions.

The Need for Supply Partnerships with Novel Purification Methods Robust supplier networks are integral in the biopharmaceutical industry, especially as demand for novel purification methods, such as high-pH technology for resins, is growing.

By working closely with suppliers and partners, drug developers and manufacturers can benefit from various advantages: 1. Access to a Reliable Supply A robust supplier network ensures a reliable supply of resins and other materials required for the manufacturing process.

Having multiple suppliers in different geographic locations and with varying manufacturing capabilities can reduce the risk of supply chain disruptions due to unforeseen circumstances, such as natural disasters, pandemics, or regulatory changes.

2. Access to the Latest Purification Technologies Working alongside established suppliers can open access to the latest purification technologies, improving the efficiency Winter 2023 Volume 6 Issue 4

Manufacturing & Processing and effectiveness of the purification process. With a partnership that provides expertise and trouble-shooting capabilities, manufacturers can produce higher-quality products faster, which will ultimately benefit patients.

3. Creating Cost Savings As profitability is becoming paramount, a robust supplier network can also help manufacturers negotiate better prices for materials and services, which can result in cost savings for the company.

This can ultimately lead to more competitive pricing for the product, which can benefit patients and increase market share.

4. Maintaining Thorough Quality Control Working with a supplier that has a rigorous quality control program can ensure that the resins used in the manufacturing process meet the required quality standards. Patient safety is imperative within the biopharma industry, and ensuring product quality is high – without any impurities or contaminants – is a key advantage of having a robust supplier network.

Exploring Off-the-shelf Resin Options and Their Advantages A robust supply chain helps to secure the immediate supply of off-the-shelf resins, which provide several advantages over custom-made resins. First and foremost, they are readily available, eliminating the need for long lead times and custom orders that can delay production timelines. This is especially important in the rapidly evolving biopharmaceutical industry, where time-to-market is critical.

In addition to availability, off-the-shelf resins offer the advantage of cost-effectiveness, as they are often less expensive than custom-made resins. They also offer consistent performance from batch to batch, ensuring the reproducibility of DSP. However, a standardised approach for implementing off-theshelf resins and innovative resin technologies into pre-approved processes is currently lacking, and regulatory approval is required before switching to alternatives.

Overcoming Regulatory Barriers when Implementing Alternative Solutions The lack of a standardised approach for implementing a new resin into a pre-approved process is a significant challenge for the biopharmaceutical industry. This has prompted the sector to come up with a regulatory-recognised solution for implementing materials from an alternative supplier or source founded on a QbD approach.

The BioPhorum guidelines suggest that a detailed documentation approach to critical material attributes (CMAs) enables interchangeability, making manufacturing integration and regulatory approval easier. To ensure consistent product quality and safety, certain controls are required, such as testing specific attributes of Protein A by the supplier, verifying the viral control strategy, and assessing DSP capacity to remove unwanted materials.

The BioPhorum convention of defining a material by its function and CMAs could allow materials to undergo an equivalency investigation instead of manufacturing revalidation, as defined in the four-step process aligned with the latest International Council for Harmonisation (ICH) guidelines: www.international-biopharma.com

1.

2.

3.

4.

Outlining the characteristics of the raw material to ensure the quality and safety of the drug product.

Definition of the attributes of the raw material, such as chemical, physical, microbial, and other safety aspects.

Creation of a summary control strategy based on the knowledge of current products and processes to ensure quality and performance.

Combination of the information from the previous steps to define the CMAs.

By following these QbD steps, as recommended by BioPhorum, supplier flexibility is increased, and it could facilitate regulatory approval processes. This is particularly beneficial when sourcing pH-sensitive products.

Looking Toward the Future As increasing numbers of novel antibody-based therapies enter the pipeline, there is a need for applying innovative technologies, a robust supply chain, and a way to incorporate these technologies into existing processes compliantly. Access to alternative purification materials is crucial for delivering new antibody therapeutics, and a QbD-led approach is essential to incorporate these materials into DSP. As more manufacturers become aware of the benefits of innovative resin technologies, adoption is likely to increase, further reinforcing the need for suppliers to continuously expand production capabilities globally.

The BioPhorum guidelines provide a regulatory-approved process for implementing alternative purification materials, enabling interchangeability, and making regulatory approval easier. A supportive supplier partnership will also be essential as these technologies increase in demand and will help to ensure consistent product quality and safety for patients.

REFERENCE 1.

https://www.globenewswire.com/en/news-release/2023/ 03/15/2627505/28124/en/Global-Bispecific-Antibodies-forCancer-Market-Report-2023-2027-Increasing-Prevalence-ofCancer-Advantages-of-Bispecific-Antibodies-Over-Monoclonal-Antibodies-and-Strong-Pipeline-D.html

Aaron Moulin Aaron Moulin received his Bachelor of Science in Biology and Ph.D. in Biochemistry, both from Brandeis University. Aaron was then a Post Doctoral Associate at Genzyme Corporation for three years, worked briefly as an Applications Scientist at Microlytic and then moved on to a Technical Sales/Project Management role at Emerald Bio/Beryllium Discover Corp.

After this, Aaron spent nearly six years at GE Healthcare/ Cytiva, first as a Support Scientist and then as a Product Specialist for Bioprocess Chromatography Resins before moving to Sartorius as a Field Applications Specialist for Chromatography instruments/hardware and media. Currently Aaron is Field Applications Specialist at Purolite supporting primarily Eastern North American where he supports the use of Praesto resins for downstream bioprocess applications.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 39

Technology

The Cloud Isn’t Everything – De-mystyfying Cloud Based LIMS Without doubt one of the hot topics in Laboratory informatics for a number of years has been the use of the cloud for hosting Laboratory Information Management Systems (LIMS) and other lab-based systems. However, is the cloud and whatever cloud model is used or recommended (i.e., Platform as a Service, Software as a Service, Infrastructure as a Service or whatever other four-letter acronym someone comes up with) really the be all and end all of lab informatics?

The Benefits of Cloud The use of the cloud to host lab informatics solutions can have real benefits. These are well documented and include changing from a capital cost model to a recurring cost model if you choose to pay on a subscription basis, the reduced need for in house IT resources to support systems and solutions, the use of third party infrastructure, automatic and guaranteed updates of operating systems and cyber security software and, potentially, automatic update of the LIMS solution as new releases become available. In addition, having a web-based solution hosted in the cloud can make it easier to manage user access and make collaboration within and between organisations simpler. The cloud can also make it easier to access data from multiple databases making it easier for data scientists to analyse large data sets from disparate sources; a key to realising the value of your data assets. However, it is always worthwhile making sure that these benefits will be of value to you, and your organisation, and any potential risks are identified and minimised.

Understand the Risks Any organisation considering a cloud-based implementation must ensure that the hosting organisation can address any concerns they may have, or risks they have identified, about third-party hosting. Reputable hosting organisations will have no difficulty doing this. However, it is worthwhile ensuring that cloud service providers have the required high levels of physical and virtual security in place and the level of guaranteed uptime should be checked. In regard to uptime, organisations must also identify the levels of uptime they need; the higher the guaranteed uptime the more it will cost, so an informed choice needs to be made. Organisations must also understand how software upgrades are applied, especially to the solution itself. Depending on the model chosen some suppliers will dictate when upgrades occur, potentially limiting the time available for users to test, validate and be trained on the new software, and forcing an upgrade on the users when it is not wanted. One other key area to check is access to your data. If you choose to discontinue your relationship with the supplier, will you be able to get access to, or a copy of, your own data and what does the supplier do with the backups and copies of your system and associated data they may hold?

40 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Consider the Business Case & User Needs In the end, however, the cloud is a delivery method for a solution and, no matter how good that delivery method is, the value of any solution lies in its ability to meet the business and data needs of the organisation and users. The key to realising the value of your data assets is ensuring that you are actually collecting the correct data and the data is consistent throughout the organisation. This data imperative must never be lost sight of. Therefore, when looking for a LIMS solution, in addition to deciding if a cloud-based solution is appropriate (and if so what flavour of cloud solution), what should you be looking for?

Many, many articles have been written about this over the years and while the basics remain the same, the inevitable changes that have occurred in the laboratory environment such as cost pressures, and workforce shortages and deskilling have brought about some alteration in these needs. The starting point for any LIMS solution must be a clear business case and defined user requirements. From here it becomes easier to select the LIMS solution that will best fit your needs and, while decisions about an on premise or cloud base solution will play a part in this, other factors must be considered.

Winter 2023 Volume 6 Issue 4

Technology

Items to consider include: •

•

•

•

Does the LIMS need to help you meet regulatory requirements such as GxP and CFR 21 Part 11 using functionality such as electronic signatures?

Do you work to defined standards such as ISO 17025 or ISO 15189 and if so how will the LIMS help you comply with aspects such as resource requirements and process requirements?

Do you need to automate your preparation and analytical workflows to guide users through them step by step, meaning you will need Laboratory Execution Systems (LES) functionality?

Are you subject to external audits, if so and if it is time consuming and difficult to find the required information, how can the LIMS help you to make this task simpler and quicker?

•

•

• • •

Do you need to integrate your instruments with a LIMS to speed up the result gathering process and eliminate transcription errors?

If you work in a manufacturing environment, do you need a LIMS to help you with traceability of raw materials and intermediate products to the final products?

Does the LIMS need to help you manage documents and document versions such as certificate of analysis?

Are your workflows and processes likely to change with time, if so, how easily can the LIMS adapt to these changes?

And the list goes on, and on

The above is just a small selection of the considerations that must be taken into account when purchasing or implementing a LIMS. Each business and user will have different priorities, concerns and requirements which will inevitably change the list. Whether your LIMS is cloud based or not is, in itself, an important decision, but despite what is suggested in some circles it is not the be all and end all of LIMS selection.

Always remember the business case, user needs, and data imperative.

Simon Wood Simon Wood PhD, Product Manager at Autoscribe Informatics, has over 30 years' experience in the commercial LIMS environment. He is an acknowledged expert in the field of scientific and laboratory informatics. With a degree in Plant Biology from Newcastle University and a PhD in Mycology from the University of Sheffield, Simon successfully moved into the field of laboratory informatics.

Email: swood@autoscribeinformatics.com

www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 41

Technology

PEER REVIEWED

The Benefits of Digital and Electrochemical Lateral Flow Assays Lateral Flow Test Basics Lateral flow tests (LFTs) are simple diagnostic technologies that allow rapid and low-cost detection of a given analyte at the point of care level. While LFAs have widespread uses including environmental testing they are more commonly associated with healthcare testing, for example the pregnancy or the SARS-COV-2 Antigen test.

LFT technology1 can be summarised as shown in Figure 1. (i) Some LFTs require extraction of the analyte (antigen) (e.g., SARS-CoV-2 Antigen) from the biological sample (e.g., mucus) in a buffer of a known pH (often at a physiological pH of 7.4) and composition (including salts and surfactants for sufficient flow/ clearance) while in other cases (e.g., pregnancy test) this is not required and the biological sample (urine) is instead directly applied to the test (ii). The sample is placed on the sample pad (which may contain buffer salts if a buffer solution is not used) whereupon it is drawn to the absorption pad by capillary action (iii). The solution passes to the conjugate pad where it dissolves antibody-gold nanoparticle (AuNP) conjugates.

The solution continues to the test line made up of unlabelled capture antibodies where, if the antigen is present, a sandwich immunocomplex forms with a second antibody coupled with AuNPs ((iv a). AuNPs are widely used and are able to reflect light in the visible region which to the observer appears as a

purple, pink or blue line when they accumulate. Conversely, if there is no antigen present, the detection antibody/AuNP cannot form a complex with the capture antibody resulting in no visible line (iv b). Next, the solution passes to the control line, where an agent specific for the detection antibody can capture it even in the absence of the analyte, so that a control line should always be visible regardless of whether the antigen is present or not. The inclusion of the control line is used to ensure that flow occurs properly, with its absence equating to a void test. (v) The solution finally passes to the absorption pad which is used to ensure consistent flow while minimising backflow.

Advantages and Disadvantages of LFTs LFT technology is well known for affording quick development cycles if the appropriate antibodies are available. This ultimately allowed many companies (e.g. Innova, Abbot Panbio) to produce LFT strips in quick response to the Coronavirus pandemic. Additionally, LFT technology is also not only inexpensive to produce (<£1) but can be mass manufactured in large quantities. This allowed governments around the world to shoulder the costs of LFTs allowing it to be used as part of the daily routine of workers during the Coronavirus pandemic. Storage of LFTs is also trivial due to their long shelf lives without requiring refrigeration allowing easier transport and their use in less developed countries. Finally, LFTs are quick to run, only requiring sample collection before carrying out the test which can take anywhere between 3–30 minutes.

Figure 1: A summary of LFA technology 42 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Winter 2023 Volume 6 Issue 4

Versatile design

intuitive delivery

Your fill volume may change, with Aidaptus® auto-adjust plunger technology your auto-injector doesn’t need to

Auto-adjust plunger technology

0.3mL - 1mL*

2.25mL

Auto-adjust plunger technology

1mL

0.5mL - 2.0mL*

Now in production Accommodates both 1mL and 2.25mL glass syringes in the same device

See our auto-adjust plunger technology in action Find out more by scanning the QR code or visiting ompharmaservices.com/ibi-dec2023

In collaboration with

*In addition to an air bubble and overfill Aidaptus® is a registered trademark of Owen Mumford Ltd, ©️2023 OMPS/ibi/ad/ob/1223/7

www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 43

Technology Ultimately, the combination of these advantages results in LFT technology being widely used at the point-of-care.

LFT technology is often contrasted with polymerase chain reaction (PCR) testing.2 PCR generally has excellent sensitivity but higher costs and the need for lab testing alongside trained scientists limit its point-of-care potential. Unlike LFA, where antigens are detected, PCR involves the detection of DNA (and RNA after conversion to DNA) through the combination of thermal based amplification and DNA polymerase enzymes. In brief, (i) the double stranded DNA helix is thermally (90–94°C) denatured revealing two single DNA strands. Next, (ii) the temperature is reduced (55–70°C), which allows short DNA sequences termed primers to bind to the ends of the single stranded DNA. In the final stage, termed elongation, (iii) the DNA polymerase enzyme individually adds free nucleotides to the single stranded DNA strand until two new double stranded DNA helices are formed.

The steps forming one cycle can be repeated until the DNA is amplified to an amount that can be detected.

Unlike detection based on the more sensitive the polymerase chain reaction (PCR), LFTs comparatively have a poor (high) limit of detection (LOD). A higher LOD can lead to the possibility of false negatives which can ultimately result in missing patients who are in fact positive. For example, during the Coronavirus pandemic, while LFTs were able to identify patients with relatively high levels of the SARS-CoV-2 antigen (typically days 3–4 after infection),3 they missed users at 2–3 days post-infection due to low antigen levels. Ultimately, while LFTs acted as an effective screening process, more sensitive techniques such as PCR were still needed to be employed to completely rule out the possibility of SARS-CoV-2 infection for those that had been in close contact to people infected with the Coronavirus. This suggests that LFTs would be more useful if their sensitivity could be improved.

Additionally, the LFT in its current form typically only provides qualitative data (i.e., positive or negative) which is

sufficient for certain cases (pregnancy/SARS-CoV-2 infection) but is not suitable for certain diseases where the analyte needs to be quantified. For example, alongside clinical symptoms, a C-reactive protein (CRP) test is used by clinicians to determine whether patients should be given antibiotics following acute bronchitis (inflammation of the lungs). Current NICE4 guidelines are as follows; CRP < 20 mg/L: do not routinely offer antibiotics, CRP 20-100 mg/L: consider a delayed antibiotic prescription, >100 mg/L: offer antibiotic therapy. Elevated CRP is also a risk for patients to develop cardiovascular based diseases with <1mg/L considered low risk, 1–3 mg/L considered an intermediate risk and >3 mg/L considered a high risk.5 Ultimately the ability for a LFT to provide a quantitative result could allow clinicians to diagnose conditions at the point-of-care.

A successful diagnosis using LFTs can also vary between users depending on experience and is ultimately inherently subjective. For example, while using the Innova SARS CoV-2 antigen LFA test it was found that trained scientists showed a successful interpretation rate of 79% but this fell to 58% when carried out by members of the general public.3 Interpretation is further complicated when carried out by users with sight difficulties reducing successful applications even further.

Ultimately, there is a clear need for LFTs to have an objective readout that removes human error.

Worryingly LFTs can also be erroneously reported or deliberately misused. School children have reportedly deliberately misused the covid LFT in order to induce a positive result (and get out of school) by applying orange juice to the LFT instead of a true sample.6 The acidic nature of orange juice results in non-specific binding of negatively charged gold nanoparticles to the test line antibodies. While adding buffer to the LFT strip returns the pH to neutral, thereby preventing non-specific binding and resulting in loss of the test line signal, it is still alarming that LFTs can be deliberately misused. Furthermore, even if the SARS-CoV-2 antigen LFT was conducted properly the user is responsible for providing and

Figure 2: An overview of digital LFA and different detection strategies 44 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Winter 2023 Volume 6 Issue 4

PYROSTAR™

Neo

Recombinant Endotoxin Detection Reagent

Convenient and Easy to Use. A new synthetic colorimetric method that can be run using a standard absorbance microplate reader.

PYROSTAR™ Neo is a new endotoxin detection reagent developed by recombinant technology.

FUJIFILM Wako Chemicals U.S.A. Corp. © FUJIFILM Wako Chemicals U.S.A. Corp. - 2023

www.wakopyrostar.com ~ wkuspyrostarinfo@fujifilm.com www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 45

Technology honest assessment of the result rather than this being done by an independent observer. The use of a fail-safe in LFT to prevent misuse alongside a non-biased way of reporting would allow greater confidence in LFTs.

taking pictures using smartphone cameras. Figure 2 provides an overview of dLFT technology alongside the different measurement modes that can be used to obtain results. An overview of the different digital LFTs is given in Figure 3.

Another limitation of the use of LFTs is that users can easily be confused if more than one analyte is being measured in a single test- in other words, if tests are multiplexed. For example, tests have been developed that can detect antigens from influenza viruses A and B as well as SARS-CoV-2 and other viruses that can infect the respiratory tract, but there is a risk that users will find these increasingly confusing and could easily misdiagnose by reading the wrong test line.

Abingdon Health have developed an absorbance-based dLFT which uses mobile phone technology as a reader.7 After light is shone at the LFT, the reader is able to determine absorbance of the AuNP by comparing incident light (light shone at sample) vs transmitted light (light detectable after passing through sample). Absorbance based measurements are typically inexpensive, yet environmental effects (e.g lighting) and variations between phone model can affect diagnosis success.

Finally, data storage is impractical or very limited with current LFT technology. The ability to store LFT data could allow the general public to have easy access to their own historical test data or even allow doctors to make clinical decisions remotely.

With effective data security in place, all of the challenges listed above can by overcome through digitisation of the technology.

Additionally, ellume have developed a digital SARS-COV-2 Ag LFT that allows digitisation based on a fluorescent readout.8 Fluorescent based measurements are similar to absorbance-based measurements but after the sample absorbs light, it emits light of a higher wavelength (lower energy) which is in turn detected.

Fluorescent based measurements have excellent sensitivity yet require specialist equipment and are more expensive.

Digital LFT Summary and Advantages Digital LFTs (dLFT) involve the integration of the standard LFT with a reader that enables analysis of the LFT to be carried out digitally rather than visually. At their simplest, this could involve

Here at éclateral,9 we have developed the first commercial electrochemical LFT (eLFT) [10] which couples an LFT with in-house electronics alongside a reader capable of carrying

Figure 3: A comparison of different diagnostic methodologies 46 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Winter 2023 Volume 6 Issue 4

Technology out electrochemical measurements. In this set-up, the label promotes a reaction that results in a product that can be detected electrochemically with an external reader. Electrochemical measurements are preferred compared to the optical techniques above because they are not subject to ambient light or the quality of different smartphone cameras, they are lower cost than fluorescent assays both of which greatly increases accessibility to patients and clinicians. Furthermore, the electrochemical nature of the measurement means that analytes can now be quantified, while the digital nature of the readout means that results can be stored and transmitted electronically. If appropriate, the test results can be immediately presented to the user or patient together with medical advice via an App. Regardless of the measurement technique, digital LFTs offer several advantages over traditional LFTs. The ability to provide a quantifiable result could allow diagnosis for conditions that were previously difficult to test at point-of-care. Importantly, analysis of the results done by an independent measurement is now objective rather than subjective, not only allowing for greater confidence in obtained results but also allowing LFT technology to be accessible for those with sight difficulties. The easy storage of data could allow personal medical records to be developed or even used on a national scale to predict disease outbreaks.

Despite this, digital LFT can have their own drawbacks especially if the use of bulky, expensive or high-maintenance readers limits their point-of-care capability and if their increased cost makes it less affordable to the general public.

Electrochemical LFTs (eLFTs) could provide additional benefits to the digital LFT readout (Figure 3) while maintaining the benefits of LFT technology previously outlined. For instance, data anonymity is a key component of double-blind trials where neither the researcher nor the patient knows the outcome of the test. eLFTs fulfil this criterion since the user is not required to view or interpret the result and eLFTs could thereby bring LFT to double blinded clinical trials where users perform testing themselves from the comfort of their home. Additionally, eLFTs could be designed to have an internal fail-safe which would limit the impact of users trying to deliberately void the test. For example, if the reaction enabling electrochemical measurement is designed to be pH sensitive, the application of orange juice leading to a signal at the test line would still lead to a void test.

Conclusion Digital healthcare is becoming increasingly important in peoples’ daily lives, allowing users to easily access their personal medical records and even allowing the ability for people to monitor their own healthcare. LFTs are inexpensive, rapid and are at the point of care level yet are limited to certain use cases due to their low sensitivity and qualitative nature. Digital LFTs take the advantages of LFTs while offering additional benefits of digitisation, namely quantification, data storage and an objective readout. Additionally, eLFTs offer further advantages such as data anonymity and an internal fail-safe compared to other digital LFTs while still maintaining accessibility to the end user due to their low cost and miniaturised format. Most importantly, the instrumentation cost of eLFTs can be up to two orders of magnitude lower compared to fluorescent readers, placing eLFTs in pole position for penetrating into the home use markets, in similar way to home-based glucose sensing devices, due to the same simple, mass manufacturable underlying technology. www.international-biopharma.com

REFERENCES 1.

2.

3.

4.

K. Koczula, A. Gallotta, Essays in Biochemistry, 2016, 60, 111-120.

Anal. Methods, 2014, 6, 333 G. Guglielmi, Nature, 2021, 590, 202-205 Scenario: Acute bronchitis, https://cks.nice.org.uk/topics/ chest-infections-adult/management/acute-bronchitis/, (accessed December 2023).

5. G. Hirschfield, M. Pepys, QJM: An International Journal of Medicine, 2003, 96, 793-807.

6. How children are spoofing Covid-19 tests with soft drinks, https:// www.bbc.com/future/article/20210705-how-children-arespoofing-covid-19-tests-with-soft-drinks, (accessed December 2023).

7. Smartphones: The lateral flow readers in your pockets, https:// www.abingdonhealth.com/smartphones-the-lateral-flowreaders-in-your-pockets/, (accessed December 2023).

8. COVID-19 Home Test, https://www.ellumehealth.com/consumers/ covid-home-test, (accessed December 2023).

9. éclateral, https://www.eclateral.com/, (accessed December 2023).

10. J. Cheng, G. Yang, J. Guo, S. Liu, and J. Guo, Analyst, 2022, 147, 554-570.

Benjamin Edwards Benjamin Edwards obtained a M.Chem at Durham University before doing a PhD in chemical biology at Imperial College London.

Here, he developed biosensors for protein biomarkers based on aptamer and nanopore technology.

After carrying out an internship for biosensors based on organic thin film transistors at NeuDrive, he joined éclateral where he works as a scientific researcher. At éclateral, Ben has contributed to two patents while working on the electrochemical methods and (bio)chemistry needed for the éclateral's technology development.

Uroš Zupančič Dr. Uroš Zupančič, CSO & Co Founder of éclateral Ltd is a co-inventor of the o~pal technology and leads the technology development at éclateral. Uroš has a PhD in biosensing technologies from the University of Bath. His previous experiences include: CSO of biotIP Ltd, visiting researcher at the Wyss Institute at Harvard University and associate scientist at Novartis.

Paul Ko Ferrigno With a background in medical and academic research (MRC Cancer Cell Unit, University of Cambridge, Leeds Institute for Molecular Medicine, former Visiting Professor, Leeds University), Paul is the CEO and Co-founder of éclateral.

He has been involved in commercialising technological innovations since 2008 when he founded Aptuscan (acquired

by Avacta Group in 2012). He is named on multiple patents, has published over 40 research papers, reviews, and book chapters, and has founded, co-founded and/or led 5 companies to date. He also invests in the future through work on multiple Boards for charitable, educational and grassroots sports organisations.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 47

Application Note

SMART First Human Dose, with PCI Pharma Services

The core concept of PCI’s SMART FHD service offering is simple: to accelerate early phase clinical manufacture and supply. In order to achieve this, PCI has taken three of its flagship services and combined them into a program that ensures a sponsor’s valuable API can get to Phase I clinical trials quicker, cheaper and more efficiently than via the traditional development route. Those three services are drug-in-capsule manufacture, using Xcelodose® micro-dosing technology; industry-leading clinical packaging services; and PCI’s bespoke clinical program management service, clinicalSMART™.

and improves data transfer, all of which contributes to greater efficiencies and speed to clinic and patient.

Service #1: Drug-in-Capsule Manufacture with Xcelodose® The traditional route through early clinical phase formulation development can be a complex and time-consuming process.

Excipient compatibility studies, prototype development, and the associated stability studies themselves can take months to complete before the product can be delivered to the clinic.

However, by providing drug in capsule (DIC) manufacturing services using Xcelodose® micro-dosing technology, sponsors can significantly reduce the time it takes to get the first-in-human dose to clinic.

Xcelodose® is a precision powder micro-dosing system that allows API to be filled directly into capsules and vials. This approach removes the need for development of a powder blend, as would be the case for a more traditional development processes. PCI operates both the Xcelodose® 120S and the 600S models, filling up to 120 and 600 capsules per hour respectively, making them ideal for clinical manufacture from early to later stage clinical programs. Additional investments in Xcelohood™ and Xceloprotect™ containment systems further enhances the safe processing of highly potent drug products at our manufacturing facility in the UK. By eliminating the requirement for pre-formulation activities, excipient compatibility and associated stability studies, as well as simplification of method development processes, Xcelodose® enables products to be manufactured and delivered to clinic up to 6 months faster than the more traditional route. Dosing directly into capsules also helps to reduce the amount of API required, helping to eliminate wastage of often-valuable APIs which is particularly important at the early stages of proof of concept if API is in short supply.

Additionally, Xcelodose® is a fully programmable system, ensuring exceptional levels of accuracy, precision and consistency whilst minimizing wastage of drug substance. Capsules can be filled with API at dosage strengths ranging from 100 µg to 100 mg and beyond, and the weight of each capsule is recorded, allowing traceability of samples that meet GMP requirements.

Enabling an optimised filling process compensates for variability in drug powder properties, simplifies method development, 48 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Service #2: Clinical Packaging Services Manufacturing DIC products containing highly potent APIs can be challenging, and not all CDMOs have the capability to package the same drug product safely on site. In 2022, PCI launched a new highly potent packaging facility (HPPF), located at the same site that hosts our Xcelodose® technology in the UK. Segregated rooms, dedicated HVAC systems, conditioned environments for packaging activities and dust extraction units to remove any airborne particulates from potent APIs ensures the highest level of operator safety and drug product integrity.

Additionally, online HAPA digital printing technology ensures that with just one reel of lidding foil, the facility is able to print artwork for different market requirements on a single packaging line, all within an envelope of very high grade control of airborne particulates. This, in turn, grants sponsors considerable flexibility and reduces waste of pre-printed materials.

This facility is a recent addition to PCI’s established global clinical packaging network. With sites in the US, Europe, the UK and Asia Pacific, and full capabilities for the primary and secondary packaging of encapsulated drug substance, PCI’s manufacturing activities are well-supported to provide FHD services to sponsors.

Winter 2023 Volume 6 Issue 4

Application Note Service #3: clinicalSMART™ PCI’s clinical Supply Management And Readiness Team (SMART) is a specialist team of Clinical Supplies industry professionals who have over 400 years’ collective experience, and who manage clinical drug supply on behalf of clients.

Since its inception in 2016, clinicalSMART™ has supported almost 100 clients and over 250 studies globally. Designed with flexibility at its core, clinicalSMART™ is able to support sponsor requirements either throughout the entire study lifecycle, from protocol development through to final destruction of materials, or at certain time points when sponsor teams require additional resources to supplement in-house supply management expertise.

It does so by collating all key information from external parties (such as trial design, drug stability, and recruitment assumptions) and utilising in-house expertise to develop optimal supply strategies. Whether it’s working with client’s internal teams in a consultative role, or assuming full ownership of supply chain management, clinicalSMART™ services are immediately available, preventing the need to recruit new staff, delivering the agreed contracted hours per month as required by the client.

Established in 2016, clinicalSMART™ addressed the industry’s growing need for integrated clinical supply management services. At that time, the two highest ranking therapeutic areas in terms of clinical trial cost per patient were haematology and oncology, with a median cost of over $200k and over $100k respectively.1 As of December 2023, there were 187,957 registered interventional clinical trials using drug or biologic therapies, up from 174,669 in September 2022.2 Considering the sheer numbers involved here, any delays, miscommunications or errors in the clinical trials management process would be extremely costly in financial terms, not to mention the most important factor: the risks to the patients themselves.

In addition to clinical supply chain management, clinicalSMART™ is also able to initiate RFQ document writing to support outsourcing requirements on a client’s behalf. After reviewing key study assumptions and estimates in order to establish an initial study plan, the RFQ and relevant supporting documentation is prepared for the client; due to a thorough knowledge and evaluation of the study requirements, a high level of accuracy within the quotation is assured. As with the study plans, RFQs are then submitted to the clinicalSMART™ team for peer review, drawing on the vast experience within the team to ensure a high level of accuracy.

Regardless of the level of CSM involvement contracted to the client, the critical objective remains the same: to provide a seamless, integrated clinical supply service at the level required by the client, at any stage or throughout the study lifecycle, whilst leveraging PCI’s extensive experience in this area of expertise.

www.international-biopharma.com

To gain maximum benefit from clinicalSMART™ it is recommended that services are contracted as early as possible, preferably prior to study start up. This enables the PCI CSM to collate and analyse key information from external study stake holders, and to review the protocol, the drug stability, the availability of comparators in the marketplace, the countries involved in the study, the labelling and distribution strategy, and recruitment projections.

This comprehensive assessment will help the clinicalSMART™ CSM identify the most efficient kit design and create an initial packaging forecast, which can be modified as changes arise.

The CSM will also ensure the forecast is ‘fit for purpose’ by assessing it against the potential drug utilisation in the IRT.

The PCI CMS will typically review the IRT specifications and recommend initial shipment triggers and re-supply values, ensuring best use of available drug product, adjusting values when live study data becomes available, and when changes arise from unplanned protocol revisions, or variance between projected vs actual site activity. This enables the PCI CSM to flag potential supplies risks, and present corrective measures.

A Combined Program: SMART FHD Services PCI’s SMART FHD service consists of a dedicated CMC development team capable of rapidly transitioning a drug product from candidate selection to first human dose clinical trials.

The ability to manufacture DIC dosage forms, a robust clinical packaging network and the expertise of PCI’s clinicalSMART™ team is a powerful combination of services which ensures that early phase drug products reach FHD clinical trials as quickly and efficiently as possible. Driving this service offering is PCI’s SMART FHD team, which consists of experienced Clinical Supply Managers, Regulatory Scientists, Formulation Scientists, Packaging Technologists, Analytical Chemists and Quality Assurance/QPs who collectively have hundreds of years of industry experience.

This experienced group will oversee all drug development preparation activities plus regulatory and clinical trial supply management, reducing clinical trial preparation from up to two years to possibly just a few months, as outlined below.

PCI’s SMART FHD team provides sponsors with a development path to FHD clinical trials that is shorter than traditional routes by a matter of several months, potentially reducing the time to market by a matter of years. The SMART FHD team achieves this by:

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 49

Application Note • •

• • • • •

Developing and manufacturing minimum and maximum DIC dosages; Qualifying test methods and conducting a bracketed stability protocol including packaging, storage, and testing, and providing reports throughout; Bottling and labeling the drug product for clinic use; Compiling all CMC information to support regulatory submission; Providing QP release of the clinical drug product; Managing the sponsor’s inventory and distributing the drug product for clinical use; Supplying project management support services to organise and oversee all aspects of the development project.

and how it may assist you in your clinical goals, please get in touch today.

REFERENCES 1.

2.

The “white glove” service that SMART FHD team provides encompasses a number of benefits to sponsors, which include, but are not limited to: • • • • • •

Rapid access to clinical data; Removal of traditional drug development from the critical path; Significant financial savings; Reduction of time delays and financial investments linked to multiple development activities; Flexibility of supply from clinical Phase I, through to Phase IIa and possibly beyond; Quality Assurance, QP, Regulatory and Analytical support from dedicated teams within PCI.

The Future of SMART FHD The value of PCI’s SMART FHD service offering is twofold.

Firstly, it enhances a handful of PCI’s existing service offerings to create a symbiotic operation for sponsors whose drug product is entering early phase clinical trials. In doing so, it displays PCI’s ongoing commitment to providing sponsors with fast, efficient services which deliver their valuable drug product to patients around the globe. SMART FHD’s continued excellence in providing seamless, integrated services aligns perfectly with the wider organisation’s goal of providing true end-to-end CDMO services for clients around the globe. If you would like to learn more about SMART FHD 50 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

https://www.statista.com/statistics/1197095/clinical-trial-costper-patient-by-therapy-area/ https://www.clinicaltrials.gov/ct2/resources/trends#TypesOf RegisteredStudies

Ed Groleau Ed Groleau, Director, Clinical Supply Chain for North America at PCI Pharma Services, has over 30 years of experience in the Pharmaceutical industry. He joined PCI Pharma Services in 2018 and became the director of his group in 2019. Ed leads the Supply Management And Readiness Team (SMART) at PCI where his team partners with clinical trial sponsors to provide any clinical supply management services from protocol development through destruction. Prior to PCI, Ed worked in numerous departments at Eli Lilly, spending 15 years in various laboratories before moving into the Clinical Trial Supplies group in 2003. In 2011 he became part of a highly integrated CM&C team responsible for overseeing the development of compounds from discovery through the proof-of-concept stage. In 2016 Ed moved to Elanco, Lilly’s animal health division, where he established a global clinical trial supplies group for developing companion and food animal projects.

Email: edward.groleau@pci.com Website: www.pci.com Email: talkfuture@pci.com

Winter 2023 Volume 6 Issue 4

March 18–20, 2024 | Barcelona, Spain

Connecting the global biopharma community to elevate life science partnerships The 18th annual BIO-Europe Spring will take place March 18–20, 2024 in Barcelona, Spain, and it will convene over 3,700 life science professionals representing more than 2,000 companies from over 60 countries. Attendees will take part in 20,000+ one-to-one meetings over the course of the event.

Produced by:

In collaboration with:

Register before January 31, 2024 and save up to €700! bioeuropespring.com

www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 51

INSPIRING SUSTAINABLE CONNECTIONS

World Forum and Leading Show for the Process Industries

10 - 14 June 2024 Frankfurt am Main, Germany #ACHEMA24

52 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

ACHEMA is the hotspot for industry experts, decision-makers and solution providers. Experience unseen technology, collaborate cross-industry and connect yourself worldwide to make an impact.

Are you ready? Join now!

Winter 2023 Volume 6 Issue 4

www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 53

BioTrinity is OBN's flagship two-day, partnering, knowledge sharing & investment conference for the life sciences industry, designed to catalyse growth for all who attend

LONDONAPRIL23-242024 Organised by

54 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

www.BioTrinity.com @BioTrinity #BioTrinity Winter 2023 Volume 6 Issue 4

www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 55

Ad Index Page 52

Achema 2024

Page 3

Aldevron LLC

Page 33

A&M STABTEST

Page 55

AngloNordic

Page 53

Arena International

Page 13

Autoscribe Informatics UK

Page 51

Bio Europe

IFC Biotech AB Page 54

BioTrinity 2024

Page 37

Catalyst Clinical Research

Page 45 & BC

FUJIFILM Wako Chemicals USA

Page 11

GenXPro GmbH

Page 28–29

Gerresheimer AG

Page 5

Novo Nordisk Pharmatech A/S

Page 43

Owen Mumford Ltd.

Page 48–50

PCI Pharma Services

Page 15

Qualogy Ltd.

Page 27

Richter-Helm Biologics GmbH & Co. Kg

IBC SGS

I hope this journal guides you progressively, through the maze of activities and changes taking place in the biopharmaceutical industry

IBI is also now active on social media.

Follow us on: www.facebook.com/Biopharmaceuticalmedia Subscribe today at www.international-biopharma.com or email info@senglobalcoms.com

56 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

www.plus.google.com/biopharmaceuticalmedia www.twitter.com/biopharmace www.biopharmaceuticalmedia.tumblr.com/

Winter 2023 Volume 6 Issue 4

1

SGS Biosafety Solutions Health Inspired, Quality Driven.

Our solutions

Supporting a variety of molecules

Our Center of Excellence for biosafety based in Glasgow provides:

Our team of experts can support manufacturers of:

• Electron microscopy • GMP Sanger sequencing • Viral vaccines Adventitious agents and species specific viruses • Replication of competent vectors • Retrovirus • Process impurities • Genetic stability • Other microbial contaminants

"Most notably, the Biosafety Center of Excellence in Glasgow has participated in the batch-testing and release of over 3 billion doses of COVID-19 vaccine, helping to increase vaccine access and bring the global population out of the pandemic."

• Monoclonal antibodies • Recombinant proteins • Viral vaccines • Cell therapies • Gene therapies

Why SGS? • More than 25 years of experience • Direct access to 155 employees, from study directors to scientists • Regulatory compliant and recognized quality systems based on cGMP/GLP • Accurate and reliable report • Fast turn around times • Online digital communication platform (booking GMP slot, protocols, results, invoices...)

Archie Lovatt, Site Manager & Scientific Director at SGS

Contact us To discuss your biosafety requirements, contact us today. +44 141 952 0022 healthscience@sgs.com sgs.com/services/biosafety sgs.com/linkedin

www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 57

The future of synthetic endotoxin detection.

PYROSTAR™

Recombinant Endotoxin Detection Reagent Limulus Amebocyte Lysate PYROSTAR™ Neo is a new endotoxin detection reagent that mirrors nature but is developed by recombinant technology.

FUJIFILM Wako Chemicals U.S.A. Corp. © FUJIFILM Wako Chemicals U.S.A. Corp. - 2023

www.wakopyrostar.com ~ wkuspyrostarinfo@fujifilm.com 58 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Winter 2023 Volume 6 Issue 4