Global Pharma Innovation - June 2019 by BLS Media

GLOBAL PHARMA

INNOVATION Issue 1 - June, 2019

A Tradition of revolutionizing healthcare world wide

One of the Largest Manufactures of stamps and dies in the world

2018 2012 Panasonic Healthcare

1966 SANYO Biomedical

With technology in the field of preservation, incubation, sterilisation and processing, PHCbi’s product range includes high-quality biomedical and laboratory equipment - backed by over fifty years of industry experience.

Preservation

Incubation

Cryogenic ULT Freezers (-150ºC) TwinGuard ULT Freezers (-86ºC) VIP ULT Freezers (-86ºC) VIP ECO ULT freezers (-86ºC) Biomedical (-30ºC/-40ºC) Freezers MPR Pharmaceutical refrigerators MBR Blood Bank refrigerators

IncuSafe CO2 Incubators IncuSafe Multigas Incubators MIR Heated Incubators MIR Cooled Incubators MLR Climatic Test Chambers

Processing BSC Biological Safety Cabinets

Nijverheidsweg 120 | 4879 AZ Etten-Leur | Netherlands T: +31 (0) 76 543 3833 | F: +31 (0) 76 541 3732

www.phchd.com/eu/biomedical

Contents - Section Headings Tableting 5 - 23

Biopharmaceutical 24 - 43

Life Sciences 44 - 45

Chromotography special feature 46 - 55

GLOBAL PHARMA | 03

Bio pharmaceutical 24 - 43

Tableting

GLOBAL PHARMA | 04

The Rise of Continuous Pharmaceutical Manufacturing Now gaining momentum in the pharmaceutical industry, continuous manufacturing (CM) presents a new approach to oral solid dosage (OSD) form production and meets the industryâ&#x20AC;&#x2122;s demands for faster product development, reduced costs and increased manufacturing flexibility

or decades, the batch production of blockbuster solid dosage forms dominated the industry. Profitability was such that companies were not incentivized to innovate or risk developing new manufacturing technology. In the post-blockbuster era, however, it is increasingly recognized that material costs during drug development are significant, new drug products are likely to be manufactured in much smaller quantities and that, for novel treatments, the development of a bespoke commercial manufacturing process is not guaranteed. Such pressures have put the costs, risks and timelines associated with traditional batch-based development and manufacturing under scrutiny. Plus, regulators are increasingly supportive of CM and manufacturers are realizing that current quality assurance costs are disproportionately large compared with other industries, wherein the production, detection and removal of out-of-specification product is vanishingly small. Providing higher yields, lower utility consumption and reduced waste, CM

is enabling drug makers to move away from stepwise and time-consuming batch processing to a fully integrated and closely controlled process that gives excellent product consistency by intrinsic design. Shifting the Paradigm Yet, despite agency support and the mounting body of evidence in favor of CM, doubts remain. Questions are frequently asked about CM’s ability to handle anything but large volumes of low-cost drugs, the finances involved in purchasing and installing appropriate equipment, whether regulators can keep up with the technical developments in continuous processing and, perhaps most poignantly, whether the conservative pharmaceutical manufacturing industry will grasp the nettle and appreciate the full benefits of “going conti.” Many companies talk about CM, but few speak with the experience of actually implementing a continuous process line. GEA’s Richard Steiner, Business Development Manager, ConsiGma®, explains why. He believes that the “large-volume,

low-cost” argument is somewhat dated. “What we’re seeing now is the exact opposite,” he says, adding: “If you look at market approvals and new launches, for example, it’s quite clear that some of the larger ethical drug manufacturers are now the front runners; they’re testing and challenging the CM business case with their legacy products and subsequently using these CM platforms to develop pipeline products and file for new drug applications (NDAs).” Regarding costs, the initial investment in any CM solution is going to be a financial challenge; but, as with the implementation of any disruptive technology, the early stages are time-, effort- and cost-intensive, including the associated organizational changes. “When, and only when we establish economies of scale, will the financial burden decline as CM equipment becomes a commodity … as opposed to a tailor-made and engineered-for-purpose solution,” adds Richard.

GLOBAL PHARMA | 06

And, in terms of regulation, Lawrence Yu, FDA’s Deputy Director for the Office of Pharmaceutical Quality, noted: “If drug makers paid more attention to high quality manufacturing, it would prevent the regulatory problems that lead to plant closures and costly fixes. Continuous processing also allows manufacturers to respond much quicker to changes in demand, potentially contributing to the prevention of drug shortages.” A key element of the US Food and Drug Administration’s Pharmaceutical Quality for the 21st Century: A Risk-Based Approach initiative, the agency understands that, although it’s not easy for drug manufacturers to transition from batch to continuous manufacturing, there are significant rewards.

scopic analysis of the powder blend and physical testing of finished product; soft sensors were used to predict critical quality attributes (CQAs). By the end of the 120-hour trial, more than 15 million tablets had been made using approximately 6200 kg of raw material in a single production area. Importantly, final analysis indicated that the campaign length could be increased even further and run for longer. Benchmarking against a typical batch process, producing the same quantity of tablets would have required 10 separate campaigns and taken a team of operators working in parallel in three production areas for 5–7 days.

terms of both time and cost. It’s all about the OEE, which influences the ROI. It’s important at this stage to highlight certain country specific differences; the arguments in favor of CM in high-cost geographies may not be valid in pharmerging economies. Nonetheless, we are seeing a great deal of interest in every major pharma market, worldwide,” he says. But, by continuing to publicize their success stories and launching more and more NDAs based on CM process technologies, Richard believes that “conti pioneers” can convince the industry that this is the future of drug production, as well as filing those products throughout global markets and supply chains, by

Continuous manufacturing (CM)is enabling drug makers to move away from stepwise and time-consuming batch processing to a fully integrated and closely controlled process that gives excellent product consistency by intrinsic design.

Real-World Proof Additionally, as the continuous processing of pharmaceuticals has moved out of the laboratory and into production environments, questions regarding the capability of the equipment to reliably and compliantly produce high quality product for long periods of time have been raised. Wishing to investigate, GEA, in collaboration with MSD, a tradename of Merck & Co., Inc. (Kenilworth, NJ, USA), recently conducted a successful robustness run on GEA’s ConsiGma® CDC 50 Continuous Direct Compression system for a period of 120 hours. The trial run was monitored using a suite of tools, including the high-frequency measurement of more than 100 process and environmental parameters, such as spectro-

Summary statistics revealed that greater than 99.5% of the production met or exceeded CQAs specifications, and less than 0.5% of the tablets were out-of-specification, providing a solid body of proof that the technology is eminently suitable for the efficient completion of campaigns that formerly involved longer and more complex batch processes. The Reality of Pharmaceutical CM

building partnerships and developing standardized manufacturing platforms that deliver faster engagement and shorter project execution times. Environmentally friendly with a much smaller footprint, CM is helping the pharmaceutical industry to produce higher quality products, enhance drug safety and reduce its industrial footprint, which provides significant advantages to governments, companies and patients alike.

Richard appreciates that operational expenditure cost savings are not actually key focus points when it comes to CM benefits. “There’s much more interest within the pharmaceutical sector in faster product development, expedited market launch and greater supply chain agility. What’s currently underestimated is the positive effect of CM on quality — in GLOBAL PHARMA | 07

“There’s much more interest within the pharmaceutical sector in faster product development, expedited market launch and greater supplychain agility. What’s currently underestimated is the positive effect of CM on quality - in terms of both time and cost. It’sall about the OEE, which influences the ROI. It’s important at this stage to highlight certain country specific differences; the arguments in favor of CM in high-cost geographies may not be valid in pharmerging economies. Nonetheless, we are seeing a great deal of interest in every major pharma market, worldwide.”

The GEA Advantage With 14 years of inspiration, GEA has firmly established its longevity in the continuous manufacturing market. Having completed more than 70 projects involving a variety of filed and authorized products - including the first ever FDA-approved breakthrough therapy developed and manufactured using the ConsiGma® platform - no other company has as much experience and done more to pioneer continuous manufacturing for the pharmaceuticalindustry. How do we do this? By making science work.

For more information Richard Steiner Business Development Manager, ConsiGma® GEA gea.com/pharma

GLOBAL PHARMA | 08

Continuous Innovation GEA and its partners are leading the way toward smaller, more

We’ve successfully completed more than 70 projects involving

flexible continuous processing technologies that have the potential

a variety of filed and authorized products, including the first

to transform the future of drug development and production —

ever FDA-approved breakthrough therapy developed and

and deliver customized quantities of drugs to patients in need in a

manufactured using the ConsiGma® platform.

quick and efficient way. The GEA Pharma Solids Center (GPSC) offers a full range Providing higher yields, lower utility consumption and reduced

of testing, development, optimization and training services

waste, continuous manufacturing is enabling drug makers to

to improve production and expedite time to market.

move away from batch production to a fully integrated and closely controlled process that gives excellent product consistency by

Contact gea.com/contact for more information

intrinsic design.

and learn how GEA is #MakingScienceWork.

More than news # 1/2019

Measuring system Ti-3 premiere in the second half of this year!

In the Tool Inspector both Ti-2 and Ti-3 we have applied highly original, innovative and unique hardware and software solutions. These devices are working in contactless mode and the measurements are made by use of the confocal sensor and the optical micrometer. User-friendly measuring procedures get the specialized knowledge unnecessary. The devices are as easy to operate as the ordinary domestic appliance.

Furthermore, by using of the convenient video system with the 5M pixels resolution, the punch pressing part optical assessment with the saving of the picture ﬁles is possible.

Ti-3

21 CFR Part 11 compliant

This function gives the possibility to compare the punch tablet pressing part conditions in the diﬀerent utilization stages and working surface details measurement like break-line or embossing.

Ti-2/3 is the system using the leading-edge technological developments in the optical micrometer and confocal sensor measurement methods. The patented measuring algorithm combining the simultaneous measurement of the synchronic movement in XY plane, enables to set down the geometrical parameters for the most complicated tablet pressing part shapes.

New in Tool Inspector Ti-3 • 21 CFR Part 11 complient • Customized data base both co-working with machine and working separately • Analysis of tooling wearing history – diagrams • Predicting the time of ﬁnal consumption • Multi-tip pairing • Multi-holes die assessment • Punches faces with protruding break-line measurement • Measurement of coated tips

www.adamus.com.pl

/adamussa

New service!

Preventive Maintenance Program

Prevention is better than cure! This also applies to machines. The wear and ageing of the tablet press components is a natural thing. Every single part of the machine has a speciﬁed durability period and is susceptible to damage. Failure, even the smallest part, can be of considerable importance for the functioning of other systems that make up the entire machine. Therefore, for the optimization of processes and proper maintenance, we recommend regular inspections and maintenance of tablet presses.

WHAT DOES PREVENTIVE MAINTENANCE GIVE YOU?

Planned inspections of machines allow to determine the technical condition of the tablet press, locate those parts to which there is a risk of failure and replace them in a timely manner. Servicing tablet presses are much cheaper than making the necessary repairs (often in emergencies it turns out that one defective element had a negative impact on the condition of others, which are also suitable for exchange), and secondly does not expose the company to additional costs related to production downtime.

Systematic reviews of tablet machines not only prevent unexpected failures or complete deterioration of a given machine but also extend its service life. By ordering regular tablet service and maintenance, you gain: • Lower failure rate • Lower repair costs • The longer working life of the tablet press • Less risk of downtime on production • Certainty of the implementation of production plans • Employee safety

www.adamus.com.pl

/adamussa

New product! Softgel capsule forms!

Softgel capsule form

We have experience and we use them

The softgel capsule is a one-piece, hermetically sealed shell wall ﬁlled with liquids. The shell protects the inner ﬁll material from atmospheric oxidation that compromises other oral dosage forms in terms of potency and shelf-life.

We support the pharmaceutical, nutraceutical, food supplement and related businesses, by manufacturing nutritional dietary supplements, and pharmaceutical products in soft gel capsules using advanced and state-of-the-art production technologies.

We know how important every detail and every millimetre of the softgel capsules shell is. That is why we have perfected a method of designing and manufacturing softgel capsule forms.

Softgel capsules advantages: • Improved bioavailability • Easier to swallow • More friendly appearance • Bigger consumer conﬁdence • Eliminates odours • Longer shelf life • Rapid action

www.adamus.com.pl

/adamussa

It’s all about turret

the most precise part of tablet press

New turret

Turret overhaul

Experience gained during a long-term cooperation with pharmaceutical industry enables us to oﬀer top quality turrets for tablet presses.

The turrets that were repaired or completely manufactured in our company ensure the tablet press operation in compatibility with the technical assumptions of their producers. We know the technical conditions that guarantee an eﬀective coupling of turrets with punches and dies.

We maintain the highest manufacturing precision and ensure appropriately selected materials, tolerances in compatibility with the requirements of tablet presses manufacturers and suitable surface smoothness. In order to increase abrasion resistance we use additional treatments of chrome plating, nitriding and nickel plating.

The understanding of the process of tablet pressing, which is necessary to manufacture high quality punches and dies, as well as our machinery park enable us to oﬀer services connected with the regaining of the correct turret functioning.

Turret basic maintenance set Our oﬀer includes: • Support in the diagnosis of turret work • Indication of turret elements in need of a repair or replacement, • Rebuilding of a whole turret or of a selected part • Manufacturing of a new turret

Turret basic maintenance – taking care of the heart of the tablet press, the most precise and the biggest part inside – responsible for tablets precise manufacturing – its condition and proper maintenance in relation to cooperating equipment: • To obtain maximum tablets output. • To minimize emergency breaks at work • To prevent damage to the tablet press • To facilitate installation and disassembly of tooling ...taking care is the cure.

www.adamus.com.pl

/adamussa

Manual polishing set

To maximize life time both punches and dies

Compression tooling surface is one of the most important, the most precise and most fragile elements in tablet compression. Tooling surface is responsible for tablets quality – their shape, appearance and weight. To obtain maximum life tablet punches and dies and minimise tablet compression problems polish your tools regularly.

Regularly Polishing: • Obtain maximum life from tablet punches and dies • Minimize tablet compression problems such as: sticking, picking, capping, tablet weight variation • Prevent damage to the tablet press • Prevent contamination of tablets In our offer we have a standard automatic drag polisher. But if you don’t have enough space or just looking for cheaper but still effective solutions, we have prepared an offer for a manual polishing set.

Manual polishing set: • Motorized polisher with foot switch to turn on the machine's operating speed • Magnifying glass with lighting • Diamond polishing paste • Magnetic holder for punch / die • Set of felt polishing pins • Microscope digital camera to measurements

www.adamus.com.pl

/adamussa

Tooling case

Customize system for storage and transportation

Don’t punch your punch! Compression tooling surface – one of the most important, the most precise and most fragile elements in tablet compression – responsible for tablets quality – their shape, appearance and weight. Based on our own experience, Adamus recommends customized system for storage and transportation for sets of punches and dies of diﬀerent standards. Furthermore, system allows to be a part of ultrasonic cleaning procedure.

• Strong heavy-duty case • Resistant to oils, water, heavy loads • Manual displacement • Storage one on top of the other • Custom made according to amount and standards tooling to co-work • Water proof case • USC bath ready-to-use

Storage

Transportation

A safe and practical storage system for punches and dies will contribute to extended life of tooling. This must be designed to ensure that tooling cannot be damaged by hitting each other and also to prevent corrosion during storage.

A custom designed trolley or tray should be used for carrying tooling from storage to the tablet press. This should include separations to prevent punches from moving/rolling around and causing damage.

www.adamus.com.pl

/adamussa

Tool Maintance Program

09. Storage With dedicated cabinets or containers,avoiding the risks resulting from mechanical damage and direct environment impact

01. Installation / uninstallation of tooling

02. Cleaning

Tooling should be cleaned to remove residuals of power and possible sintered product residues

08.

For longer period of storing, tooling shall be protected from moisture and aggressive environments

03. I

Tool Maintenance Program

Optical assessment of working surfaces of the tooling

04. Repair When minor disturbances were found

07. Measuring

Required to ensure dimensions quality of tooling before use or before storing to give a time to order new set

05. Polishing 06. Re-cleaning

After repairing and polishing, re-cleaning to remove any residual of polishing/repairing agents

Giving the work surface a smoothness to avoid common tableting problems

www.adamus.com.pl

/ adamussa

About ADAMAS S.A. The company provides its clients with competitively priced products and services of the highest quality and maintains short order delivery dates at the same time. ADAMUS S.A. is a leading supplier of compression tooling and spare parts for tablet presses, capsule fillers and blister lines for the pharmaceutical industry. With nearly 40 years of experience in designing and manufacturing tools and machine components used in various industrial sectors, ADAMUS S.A. continually delivers world class support and customer service, being a leader on the market of technological solutions of the highest quality and competitive price. A modern machinery park with its own heat treatment facility, robotic production lines, a restrictive Quality Management System and an extensive sales network covering over 55 countries, on six continents, allows us to be recognised as one of the largest and well respected world supplier of this type of products. Our Engineering Office operates with modern CAD / CAM system, 100% final quality control and ISO 9001: 2018 Quality Management System. We guarantee products and services of the highest quality, still at a competitive price. We know how to meet high demands and satisfy the needs of our clients, partners and customers on the global market. We know how important your trust is. We focus on development, continuous improvement and technology transfer. We are continuously developing our products portfolio to be able to satisfy all needs of our customers.

Our product portfolio consists of: • punches and dies of different shapes for tablets pressing • spare parts for tableting machines (tablets pressing machines) as complete rotors, rolls, cams etc. • spare and formatting parts for the blister machines (tablets packaging) • spare parts for capsule machines • punching dies, press – forming segments • other precise parts for machines and installations according to the customer’s individual order • ultrasonic cleaners • computerized measuring tools • single station tablet presses • storing solution for tools. In ADAMUS S.A. we care for harmonious coexistence with the natural environment, undertaking numerous activities that minimize the impact of our manufacturing process on the environment. We feel co-responsible not only for the immediate environment of the company, regarding the dimension of natural resources, but we are also aware that the global network of manufacturers regarding ecological standards, is as strong as its weakest link. Bearing this in mind, at every stage of production, maintenance, logistics or utilization we care about the environment, still minimizing the negative impact on the environment. ADAMUS S.A. commitment to sustainable development is reflected in such activities as: • • • • • •

using and implementing advanced environment-friendly technologies constant monitoring and minimizing pollutant emissions monitoring our industrial processes and their impact on environment making prudent use of raw materials promoting pro-ecological education and eco-friendly behaviours development and use of reusable packaging for transport and storage of manufactured components. GLOBAL PHARMA | 18

The Application & Advantages of Extended Head Flats for Tablet Compression Tooling By: Kevin Queensen, Natoli Engineering

The Rise of Continuous

A standard head flat vs. an extended head flat on a B-type punch.

All too often, tablet manufacturers work with products that are difficult to compress. When these situations arise tablet manufactures may explore a variety of options to compress a viable tablet, including adjustments to the tablet compression tooling. Fortunately, a variety of tooling modifications are available to help successfully manufacture difficult-to-compress formulations. Tool coatings or alternate steel compositions are frequently considered, however, one commonly overlooked punch modification to enhance the successful tableting of a difficult product is utilizing extended head flats. An extended head flat increases the diameter of the flat area on top of the punch head providing a longer dwell time. Dwell time is the amount of time the head flat spends in contact with the pressure roll and thus the time the punches are compacting the powder into a tablet. Dwell time is dependent on press speed, the pitch circle diameter of the turret, and the head flatâ&#x20AC;&#x2122;s diameter. Extended head flats may compact poorly compressible products better as a result of the increased compaction time and may even reduce the amount of force required to attain a specific tablet hardness. An additional benefit is that extended head flat punches typically do not require any modifications to the press and can be used with cam tracks that meet Tableting Specification Manual (TSM) or European Union (EU) standards. Increasing the diameter of the head flat is the easiest way to prolong dwell time without switching to a press with a smaller turret pitch circle and fewer stations, or, without decreasing the turret speed, both of which would decrease production. For example, a Fette 2090 press has a pitch-circle diameter of 410 millimeters, and assuming an operating speed of 50 rpm with a standard B-type punch and TSM domed head flat of 9.525mm (0.375 in) would have a dwell time of 8.88 milliseconds. Meanwhile, on that same press, a B-type punch with TSM domed head with an extended head flat (15.0mm, 0.591in) would have a dwell time of 13.98 milliseconds (Figure 2). Figure 3 illustrates the differences in dimensions between a standard B-type, TSM domed head and a Natoli B-type, TSM domed head with an extended head flat punch. The larger head flat increases dwell time by more than 50 percent without reducing the turret speed.

Figure 1

Figure 2

Figure 3

GLOBAL PHARMA | 20

The extended head flat punch can also reduce the amount of compression force needed to form a tablet at a given breaking force, also known as hardness. Tablet hardness and density are related to both compression force and dwell time. If the amount of time spent under compression increases, the amount of force necessary to maintain the same tablet hardness may decrease. The reduction in required compression force depends on the characteristics of the granulation being compressed. Because the required compression force is dependent on the product being compressed, it’s impossible to predict accurately how much off an increase in dwell time will decrease compression force. The

effect of dwell time on compression force can best be quantified during the research and development stage and understanding the relationship of dwell time to compression force can mitigate issues that commonly arise during scale-up to large production presses. Geometrically, the design of the extended head flat differs only slightly from a TSM domed head or an EU head. Other than the increased head flat, one notable difference is a reduction in the head’s thickness to allow the larger flat to fit through the same cam profile as that of a standard TSM domed or EU head. It’s this slightly thinner head that allows extended

head flats to traverse the standard cams without modifications to the cam tracks or press. The head flat can even be customized to achieve a specific dwell time on a given press. The limiting factor in the head flat’s size is the neck diameter of the punch. The neck transfers the force from the head to the barrel and tip and then to the granulation. As you can see in Figure 4, if the head flat gets larger than the neck’s diameter, it won’t have the support required to transfer the force generated when it contacts the pressure roll, which can cause the head or neck to fail.

Figure 4

With 14 years of inspiration, GEA has firmly established its longevity in the continuous manufacturing market. And having completed more than 70 projects involving a variety of filed and authorized products, including the first ever FDA-approved breakthrough therapy developed and manufactured using the ConsiGma® platform, no other company as much experience and done more to pioneer continuous manufacturing for the pharmaceutical industry. How do we do this? By making science work.

Extended head flats can also be oval or elliptically shaped. Although this design extends the dwell time exactly as a round extended head flat does, it has some drawbacks. The primary drawback is that the oval head flat can pass under the pressure rolls only in one direction along the major

axis of the oval or ellipse to extend dwell time. For that reason, punches with an oval head flat must be keyed on the upper AND lower punches, even when round, to prevent them from rotating as they pass under or over the pressure rolls. With upper and lower punches requiring keys for

oval or elliptical head flats, punch heads can experience accelerated wear because of repeated contact between the punch head and the compression roller at the same spot and no punch rotation.

GLOBAL PHARMA | 21

Additionally, if a turret doesn’t have lower key slots, then round tooling with an oval head cannot be used because the benefit of the extended head flat will be lost if the lower punch rotates. Another limitation is that if two presses have turrets with key slots of different angles, you can’t interchange the oval head flat’s punches because the different angle will alter the orientation of the head flat to the pressure rolls. Punch head profiles and the resulting dwell time play an essential role in the compaction characteristics of many drug products. Using extended head flat punches can provide a quick and reliable way to increase dwell times and, in some cases, reduce compression force without the need to modify the tablet press or cam tracks. Round, extended head flat punches don’t require keyed tooling for round shapes, as oval ones do, and that allows them to be used on many makes and models of tablet presses interchangeably.

Although extending the head flat is beneficial in many situations, it is important to note that extended head flats and the resulting extended dwell time is not the solution to all tableting issues. In some cases, the reduced or eliminated head flats have been shown to reduce ejection forces and can be beneficial in solving capping issues.

tooling vendor like Natoli Engineering early in the development process can help minimize compression-related issues that could ultimately limit production. Considering the role of dwell time in the R&D stages of product development will allow for the determination of which head flat (standard, extended, or even reduced/eliminated) is ideal for specific products.

Recent studies, conducted by Natoli Engineering, have shown that for some products reducing the head flat or even eliminating the head flat entirely (thus reducing or eliminating the dwell time) can be beneficial in some circumstances. Working with a knowledgeable

Kevin Queensen is a mechanical engineer and works as a technical service support specialist for Natoli Engineering in St. Charles, MO. Natoli is the global leader in the manufacturing of tablet compression tooling, and a leading provider of tablet presses, tablet press replacement parts and accessories, tooling inspection systems, and encapsulation change and spare parts. Natoli also provides customized consulting services, tablet or tooling design, tablet press refurbishment, analytical and operating system software, and technical training.

GLOBAL PHARMA | 22

QUALI T Y

IS NOT AN ACT,

BUT A HABIT. ~ Aristotle

NATOLI ENGINEERING COMPANY, INC.

natoli.com • info@natoli.com • +1 636.926.8900

Bio pharmaceutical 24 - 43

Biopharmaceutical

GLOBAL PHARMA | 24

One Trusted Source for Stability Storage Services Source BioScience has been providing outsourced controlled environment and stability storage from centres in the USA, Europe and the UK, to the pharmaceutical, food, cosmetics and chemicals industries, for many years. Clients receive a precise, condition-controlled storage service that ensures that by the time products reach the end-user, they are storage-safe, irrespective of external environmental factors.

Extensive GMP-compliant equipment and facilities meet rigorous ICH standards, as well as being able to be adapted to deliver unique bespoke conditions. Pharmaceutical products, clinical trial material, pharmaceutical bulk chemicals plus anything required upon request can be stored in certified and monitored temperature and humidity controlled conditions with back-up emergency systems in place. Quality & Compliance At Source Stability Services we maintain an extensive Quality Management System to control and manage every aspect of our sample storage and management service. Our documentation is the core of our client service guarantee whilst maintaining compliance with regulatory agency requirements. They also ensure our outsourced solution integrates with your existing business requirements. All our documentation is verified by our Quality Assurance onsite team.

requirements. This includes intermediate testing and accelerated testing per ICH Q1A (R2). Controlled Drug Storage GMP compliant, DEA Schedule I-V storage and controlled drug storage are offered for all ICH and custom conditions, and subject to additional security and monitoring measures. 52 m3 walk-in rooms come as standard but as Source Bioscience’s manufactures its own storage equipment, the facilities can accommodate any capacity. This means there is no limitation on the size or volume of product that can be stored. Medical Devices In healthcare and medical environments, high performance chilled equipment is essential for keeping sensitive, delicate material within meticulously controlled storage parameters. Photostability

Pharma Product Shelf Life Testing Source Stability Services offers state of the art environmentally controlled and monitored stability storage facilities to meet all ICH requirements. Our GMP facility contains rooms and chambers that have been mapped and validated for long- and short-term shelf life studies under various temperature and humidity

Photostability testing is an integral part of stress testing, recommended by The ICH Harmonised Tripartite Guideline covering stability testing of new drugs and drug products. Source BioScience offers photostability testing using cabinets that have been specifically designed to meet ICH Q1B standards to ensure your photostability studies are reliable, accurate and stress-free. Stability Storage environmental

products. With a track record in the design and manufacture of ICH-compliant and bespoke environmental controlled storage chambers, Source Bioscience offers a full complement of controlled environment products, stability storage cabinets, reach in chambers and walk in rooms to meet any need, whether large or small.

• Stability Storage Walk-In Rooms • Stability Storage Reach-In Rooms • Stability Storage Cabinets • Laboratory Chilled Equipment • Laboratory Incubators • Textile Conditioning • Data Loggers and Chart Readers • Service and Validation • Blood Banks To find out more please visit www.sourcebioscience.com, email sales@sourcebioscience.com or call +44 (0)115 973 9012

GLOBAL PHARMA | 25

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Delivering multiple benefits in medicines formulation The taste of a medicine is an important compliance factor for certain patient groups, including children, the elderly, those with chronic conditions and even in the growing field of veterinary medicine. Choosing the right filler-binder can enhance overall palatability significantly. With galenIQ™ the German company BENEO offers an excipient that can achieve this in a wide range of applications.

Sugar-like sweetness galenIQ™ is the pharmaceutical grade of BENEO’s ISOMALT, a disaccharide alcohol derived from beet sugar. Its source material is the main reason why galenIQ™ has a sweetness and taste profile that is very close to sucrose. With its well-balanced sweetness, the filler-binder has no significant off-tastes or aftertaste. galenIQ™ reduces the bitter taste of APIs, masks the unpleasant taste of ingredients such as plant extracts and contributes to a palatable pleasant taste profile and mouthfeel in the final pharmaceutical or nutraceutical product. A recent comparative study has shown that galenIQ™ is able to considerably suppress the bitterness of a quinine hydrochloride solution. In comparison to maltitol, mannitol and sucrose, the addition of galenIQ ™ (grade 721) in concentrations as low as 3 to 6% w/w led to a significantly higher reduction in bitterness. It is therefore not surprising that galenIQ™ is the excipient of choice where taste matters. A range of grades galenIQ™ is primarily considered to be a soluble filler-binder for tablets and powdered products. However, the past 10 years have proven it to be

a truly multi-functional excipient: It has been used to coat solid dosage forms, as a component in hot melt extrusion processes and, most importantly, it has improved the taste of many medicines, even in liquid applications.

application, it is important that bulk excipients fulfil the necessary requirements, such as excellent flowability, low hygroscopicity, high physical stability during mixing and high dilution potential and content uniformity, to name just a few.

The galenIQ™ product range comprises different grades that serve a broad variety of dosage forms. Besides the agglomerated grades 720 and 721 for direct compression and dry blend applications, the 800 series offers special powder grades of different solubilities and particle size distributions for wet granulation, roller compaction and other agglomeration processes.

Technological benefits With well-defined particle size distribution, galenIQ™ 720 and 721 provide outstanding flow and mixing properties. galenIQ™ is the perfect excipient for easy tableting because of its excellent compactability and high dilution potential. At the same time, only low compaction forces are required to achieve a high tablet hardness. Agglomerated galenIQ™ is a white, odourless and water soluble material with a unique morphology. The porous and large surface areas enable high concentrations of active ingredients to be incorporated without compromising the flow properties of the final mixture.

The sachet trend Due to its cost effectiveness and time saving benefits, dry blending of all ingredients without further processing is the most favoured method of drug preparation. Dry blends can be used in compression and compaction processes or simply filled into capsules, bottles, or sachets. The “sachet trend” is already becoming evident, with line extensions of well-established brands increasingly moving into sachets and stick pack packaging. Dry blends facilitate a convenient and fashionable form of drug delivery. When developing these powder mixtures for oral

Regulatory approvals Isomalt (galenIQ™) is monographed in leading pharmacopoeias (USP-NF, Ph. Eur., BP) and is approved in both Japan (JP) and China (with an Import Drug License). Source: Luhn, Habara, Cepok, Black and Bernard, PharmaChem, 9-10, 2014

GLOBAL PHARMA | 27

www.BENEO.com

The filler-binder excipient that makes medicine taste better BENEO GmbH, part of the Südzucker Group, offers galenIQTM (Isomalt), which is a water-soluble pharmaceutical excipient filler-binder, derived from sucrose. In fact galenIQ™ also has a sweetness and taste profile that is very similar to sucrose and therefore galenIQ™ is frequently used to improve the palatability of bitter-tasting active pharmaceutical ingredients (APIs), plant extracts and probiotics. galenIQ™ is non-cariogenic and shows a low glycaemic index which makes it the optimal choice for the formulation of a broad variety of dosage forms, such as chewable tablets, compressed lozenges, oro-dispersible mini-tablets, effervescents, and cough syrups BENEO is a division of the Südzucker Group, which employs more than 1,000 people and has production units in Belgium, Chile, Germany and Italy. For further information please contact: Dr. Maj-Britt Cepok, Product Manager PHARMA galenIQ Maximilianstraße 10, 68165 Mannheim, Germany Phone: +49 621 421 170 Fax: +49 621 421 160 Email: maj-britt.cepok@beneo.com GLOBAL PHARMA | 28

High-content screening of complex physiologically-relevant cell models High-Content Screening (HCS) approaches have become increasingly common in drug discovery with HCS technologies integrated across the drug discovery pipeline, from target identification/validation to high-throughput phenotypic screening to lead candidate drug characterization including SAR, MOA, and toxicity profiling. Recent years have seen an increasing demand for these drug discovery and development processes to use more predictive, higher complexity, physiologically-relevant threedimensional (3D) cell models which better mimic in vivo environments than simpler two-dimensional (2D) models. By reproducing important parameters of the in vivo environment, 3D models can enable improved assessment of drug toxicity and target validation. Such 3D cellular models may include spheroids, organoids, or induced pluripotent stem cell (iPSC)-derived cell models. Another important and expanding field for 3D cell culture-based models is that of ‘organ-on-a-chip’, whereby living human cells are assembled onto microchips using microfluidic

technologies. This technology has the potential to revolutionize drug development, disease modeling, and personalized medicine. These microchips offer an alternative to traditional animal testing and can potentially offer a quicker path to clinical trials. While the development of quantitative assays using 3D cell models has emerged as an attractive investigative tool, challenging 3D high-content image acquisition and analysis workflows have hindered wider adoption by the screening and automated imaging communities. Next-generation high-content, high-throughput tools for microscopy offer innovative and automated techniques for evaluating this complex biology. One company that

has led the way with this technology is Molecular Devices. With technology such as the ImageXpress® Micro Confocal High-Content Imaging System and MetaXpress® 3D Analysis Module with 3D Viewer, screening 3D cell models within a single, integrated interface can dramatically reduce the time to discovery. Implementation of these more complex 3D assays also requires high resolution to capture publication-quality images and data. Enhanced assay sensitivity can be achieved by taking advantage of the optical properties of confocal imaging, capturing images with a high signal-to-noise ratio while reducing out-of-focus light for crisper images and accurate cellular detail.

GLOBAL PHARMA | 29

Molecular Devices is partnering with companies who specialize in physiologically-relevant cell models such as MIMETAS, who offer the OrganoPlate®, a unique 3D organ-on-a-chip platform. This is a fully compatible microfluidic culture plate, enabling testing of compounds in any throughput on miniaturized organ models. Molecular Devices also work closely with HCS Pharma, who create innovative 3D cellular models which enable researchers to perform phenotypic screening on more relevant 3D cellular assays that also consider the extracellular matrix (ECM). To reach this goal, they have acquired BIOMIMESYS® technology, which is a unique and natural hyaluronic acid-based hydroscaffold, biofunctionalized with other ECM components to better mimic the microenvironment of every organ! All of these technologies are being imaged and analyzed with the ImageXpress® Micro Confocal High-Content Imaging System. There is also is a trend towards simplicity in high-content screening approaches. Researchers create the greatest value for their organization

when they focus their efforts and time on their research and not on learning how to use the suite of complex instrumentation within their laboratory. With this in mind, the ImageXpress® Pico Automated Cell Imager was recently launched as an easy-to-use imaging system with over 25 pre-configured analysis protocols available. In addition, it features a browser-based, icon-driven software which enables untrained scientists to easily access their data anytime and anywhere.

Molecular Devices’ HCS solutions do offer some unique capabilities that may help reduce the overall “data burden” One of the challenges with the increasing complexity of HCS approaches, particularly with the

movement towards complex 3D models, and the resurgence of multi-parametric phenotypic screening, is the increased volumes of both image data and metadata, which needs to be stored and archived safely, resulting in greater reliance on a high-quality, redundant IT infrastructure. This challenge is further compounded by the necessity to conserve data for the long term. Finally, while cloud-based data management has been discussed as a possible solution, at this point such infrastructure is unlikely to adequately address the demands of these higher-complexity assays, or to overcome the reluctance of users to store their data on third-party servers. That said, Molecular Devices’ HCS solutions do offer some unique capabilities that may help reduce the overall “data burden” that comes with adoption of these more complex screening approaches. For example, they have developed targeted imaging workflows to pre-scan samples at low magnification in order to identify hits, and then capture more detailed data for only those hits at higher magnification – ultimately streamlining hit evaluation and saving disk space.

Article contributed by

Andy Bashford, Phd

European Imaging Application Scientist, Molecular Devices Andy Bashford works as part of the cellular Imaging team with practical experience in 2D and 3D cell culture techniques. He has spent time characterizing cell lines and teaching Good Cell Culture Practice as part of the European Collection of Authenticated Cell Cultures. During his PhD at the University of Bath he used immunohistochemistry and 3D neurosphere models to investigate the development of the pre- and perinatal mouse brain.

www.moleculardevices.com +44 1189 448 000

GLOBAL PHARMA | 30

A Sterile Work Path by Douglas Watts

Douglas Watts is a qualified electronics and mechanical engineer, journalist and public relations professional of over 40 years’ standing and is proprietor of PR Options.

utoclaves are borne out of a necessity to decontaminate. This could be making waste safe ready for ultimate disposal, or the routine sterilisation, before and after use, of instruments, petri dishes and glassware used in lab processes such as culture grow. Whatever the application, the requirement is clearly defined – that of making items ‘safe and pure’. Laboratory autoclaves are a familiar sight in most research university labs and clean rooms and often taken for granted, despite the importance of their role in protecting the environment and preventing spread of disease and infection. They are generally available in a number of shapes, sizes and configurations, all with the same operating principle of using saturated pressurised steam to kill potential harmful bacteria, particularly in bio-hazardous waste. Maintaining Lab Sterility Every lab is unique in what processes and procedures take place – some requiring a more sterile working environment than others. There may even be airlocks for staff to pass through, extracting, cleansing and renewing the air, a system in operation where high-risk conditions are the norm. In a similar manner, some laboratories need to have a secure, clean exit path for its waste media so that it can be safely discarded. To satisfy this requirement, some prominent laboratory autoclave manufacturers such as Priorclave are able to build pass-through (also referred to as double-door) steam sterilisers. These create a sterile path in and out of sealed laboratories for waste. In general terms, this type of autoclave is employed within clean rooms and containment suites. In the case of a clean room application, the autoclave is used for the sterilisation

of equipment entering a clean or aseptic area such as a pharmaceutical production environment. In a containment application, the autoclave is used for the de-contamination of material prior to its release from the containment suite which would typically be a laboratory handling high-risk hazardous material. In both cases, isolation of both ends of the autoclave is required. In the case of a containment suite, it is usual for most of the autoclave to be located in the unloading room with just the door section of the autoclave protruding into the containment area. This enables most maintenance tasks to be completed without the need for an engineer to enter the containment area. For a clean room installation, it is usual for most of the autoclave to be located within the loading room, as this enables maintenance tasks to be performed without the need for the engineer to enter the clean area. These autoclaves require inclusion of a bulk head, a unique fitment enabling the autoclave to be built into a dividing wall separating the lab from the outside world. Cat 3 Lab Another autoclave requirement is to satisfy the needs of any organisations looking to create a Cat3 lab. Advan tages to designated CAT 3 application areas include much easier access beneath the autoclave, allowing cleaning and disinfection of the work

surface to prevent spread of virus and eliminate potential cross contamination. One of the first laboratory autoclaves to come onto the market to meet this requirement was designed and built by a British company – Priorclave Ltd. It was their Compact C40 vacuum autoclave, a versatile wide-format, front-loading 40L machine with stand-off legs Conclusion To ensure the sterility of a busy laboratory a number of factors must be considered to maintain a safe working environment. There is no perfect off-the-shelf answer since every laboratory, every application and every location will have unique requirements and goals; it is always best to seek expert help from autoclave manufacturers before making a major investment for your lab. GLOBAL PHARMA | 31

Anti-Idiotypic Antibodies - Essential Tools for Immunogenicity Assays Anti-idiotypic antibodies (anti-IDs) are becoming increasingly important for preclinical and clinical research. Their versatile application possibilities make them the optimal tools to support the development of new therapeutic antibodies and biosimilars. Here, we will briefly describe the different properties anti-IDs can have and how they can contribute to build a reliable immunogenicity assay. What is an anti-ID? Anti-IDs are directed against the variable region of another antibody, which contains the antigen-binding site (paratope). This is possible because the variable region of antibodies, in turn, comprises antigenic determinants (idiotopes) itself. Collectively, idiotopes are referred to as â&#x20AC;&#x153;idiotypeâ&#x20AC;? (ID), and defined as the unique proportion of antigenic determinants of the variable part of an antibody. In general, idiotopes are clonally unique to antibodies, thus anti-IDs are specific to only one particular antibody. Since some idiotopes lie within the paratope while others are close but outside the antigen-binding site, anti-IDs can be classified depending on their binding modes and specific properties into the following subgroups: GLOBAL PHARMA | 32

Development of Positive Control Antibodies for Immunogenicity ELISAs Positive controls in immunogenicity assays should represent the whole spectrum of anti-drug antibodies (ADAs) to a biotherapeutic product, as they occur during unwanted immune reactions after treatment in patients. Ideally, a positive control should be a human antibody preparation, which is usually not available in sufficient quantity for continued use. Instead, animal serum raised against the biotherapeutic product, or monoclonal anti-IDs can be used. Polyclonal anti-IDs When considering polyclonal anti-ID preparations as positive controls for immunogenicity assays, it should be taken into account that in various animal species, the immune response to a therapeutic monoclonal antibody (mAb) may not actually reflect the immune response caused by the same drug in humans. Since nowadays many therapeutic mAbs are humanized or fully human, ADAs in humans are mainly directed against epitopes within the variable

complementarity-determining regions (CDRs) of the mAb. By contrast, the hyperimmunization of non-primate animal species used for polyclonal antibody generation may lead to substantial antibody generation against epitopes of the constant regions. Consequently, the FDA recommends that i) “positive control antibodies generated by immunizing animals be affinity purified using the therapeutic protein product”, and ii) “for therapeutic mAb, the sponsor should select a positive control antibody that binds to the variable region of the therapeutic mAb” (FDA Guidance). To achieve this, an elaborate two-step purification procedure of polyclonal anti-ID preparations should be considered. Therefore, affinity purification against the biotherapeutic product is followed by the subsequent depletion of anti-human-IgG antibodies. Monoclonal anti-IDs In general, monoclonal anti-IDs are used as detecting reagents in pharmacokinetic (PK) and pharmacody-

namic (PD) ELISAs. Monoclonal anti-IDs, individual or panels, which bind to the variable region of a therapeutic mAb, may also be used as positive controls in immunogenicity assays. They can be generated in fully human antibody formats and have the advantage of the time-consuming affinity purification being redundant. In addition, monoclonal anti-IDs provide reproducible results, and can be produced unlimited. However, it should be considered that individual monoclonal anti-IDs might not adequately represent the entire range of ADAs as occurring during unwanted immune response. In summary, the setup of reliable immunogenicity assays largely depends on the careful selection and characterization of a positive control. To this end, both polyclonal and monoclonal anti-IDs are useful tools and their choice should be considered thoroughly. Author: Dr. Marieke Tesch, Marketing / Sales Manager BioGenes GmbH, Berlin, Germany Contact: marieke.tesch@biogenes.de GLOBAL PHARMA | 33

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Gilson Announces DISPENSMAN, An Accurate and Safe Bottle-top Dispenser DISPENSMAN™ Provides Accurate and Safe Bottle-top Dispensing of Many Common Laboratory Liquids Gilson, an industry leader in liquid handling, purification, and extraction systems, today introduced DISPENSMAN™, a bottle-top dispenser for convenient, safe, and precise delivery of common laboratory solutions and solvents. DISPENSMAN continues the innovation of Gilson’s liquid handling line, which also includes PIPETMAN®, the first continuously adjustable-volume pipette. DISPENSMAN has an innovative, three-position nozzle that controls

the dispensing of liquids, the removal of air in the system to avoid liquid loss, and includes an anti-drip safety feature that returns liquid to the bottle. Make volume changes quickly and easily by pushing to unlock and then sliding the volume adjuster to the desired dispensing volume with no need to open bottles, decreasing chances of contamination. DISPENSMAN is easily calibrated by the user and accommodates most liquids including many acids, bases, and solvents. The system is autoclav-

able, and does not require consumables such as tips. Five DISPENSMAN models are available that cover various volume ranges from as low as 0.25 mL up to 50 mL. All meet ISO 8655 specifications. A variety of adaptors are also available that allow the DISPENSMAN to be used with most common bottle types. For more information on DISPENSMAN bottle-top dispenser, please visit www.gilson.com.

About Gilson Gilson is a family-owned global manufacturer of liquid handling, purification and extraction solutions for the life sciences industry. We help researchers advance the pace of discovery by creating easy-to-use lab instruments that improve reproducibility and traceability. Since 1957, we’ve been developing innovative products such as PIPETMAN®. By partnering closely with the scientific community, we’re continuously advancing our product offerings and have added automated pipetting systems and software to our portfolio. Backed by worldwide R&D, service, and support, Gilson strives to enable verifiable science and make lab life easier for our customers.

GLOBAL PHARMA | 35

New Benchtop Instrument Line Improves Lab Efficiency and Productivity

About Gilson

Middleton, WI (January 14, 2019) - Gilson announced today the launch of a new benchtop instrumentation product line offering mixing, temperature control, and centrifugation of samples. The instruments feature advanced technologies that improve lab productivity and save valuable time. In addition, the digital controls on many of these instruments offer adjustable speeds and temperatures, providing increased flexibility and accuracy that allows them to be used across a variety of workflows and techniques. The line complements Gilson purification, extraction, and liquid handling product offerings, where they work harmoniously in a variety of lab workflows with existing instruments. Backed by Gilson’s reputation for reliability, the new benchtop instrumentation line provides cost-effective solutions that improve lab efficiency and accuracy. The initial benchtop offering includes the CENTRY™ 101 Plate Centrifuge, the CENTRY™ 117 Microcentrifuge, the Digital Mini Incubator, the Digital Drybath series, the Mini Vortex, the Vortex Mixer, the Digital Hotplate Stirrer, and the Roto-Mini Plus. These new instruments have small footprints, allowing them to easily fit on the most crowded benchtops. For more information on Gilson’s new benchtop instruments, please view the range here

GLOBAL PHARMA | 36

The ASPEC® Positive Pressure Manifold for Solid-Phase Extraction Offers More Consistent Flow Control for Improved Reproducibility Middleton, WI (DATE) – Gilson, an industry leader in liquid handling instruments, announces the introduction of the ASPEC® Positive Pressure Manifold for solid-phase extraction (SPE). The ASPEC manifold offers an initial step to SPE automation for clinical, forensic and food or beverage testing laboratories. With the ability to accommodate 96-well SPE plates, the ASPEC manifold is also an ideal choice for sample cleanup prior to LC-MS for bioanalyses . The ASPEC Positive Pressure Manifold uses positive pressure gas to force viscous samples into SPE cartridges, unlike traditional SPE vacuum manifolds. The multiple adjustable flow rates improve consistency among samples and recoveries, and ensures uniform flow for both low and high viscosity solutions. The ASPEC Positive Pressure Manifold, with its single gas input and small footprint, incorporates easily into laboratory workflows and provides a fast and simple way to prepare several samples in parallel for later analysis by chromatography. The universal design of the ASPEC Positive Pressure Manifold makes it compatible with popular formats, including 1, 3, and 6 mL SPE tabbed and tabless cartridges and 96-well SPE plates. When combined with Gilson’s PLATEMASTER® microplate pipetting device, the ASPEC Manifold greatly increases sample preparation throughput to meet increased testing demands in the clinical, forensic and food or beverage laboratories. For more information, visit www.gilson.com.

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The VERITY® 1910 MS Detector Offers Expanded Mass Range for Detection of Peptides and Small Proteins Middleton, WI (March 19, 2019) - Gilson announced today the launch of the VERITY® 1910 MS Detector, offering numerous enhancements over its popular VERITY 1900 Detector, including an expanded mass range of 50–1400 m/z. This broader mass range addresses the detection needs of many pharmaceutical and biotech scientists performing preparative chromatography purifications on peptides and small proteins. The enhanced VERITY 1910 MS Detector allows users to collect samples based on target mass, which saves time by decreasing the overall number of fractions that require further processing and analysis. The detector has the ability to collect full scan signals and up to four selected ion monitoring (SIM) channels simultaneously. The VERITY 1910 MS Detector relies on chip-based technology, which decreases ownership costs through reduced maintenance (e.g., changing rough pump oil). Compared to traditional single quadrupole mass spectrometers, the VERITY 1910 MS Detector offers exceptionally quiet operation with significantly less heat generation. These benefits, along with its small footprint, make the VERITY 1910 MS Detector adaptable to a variety of laboratory environments.

“Besides an expanded mass range, the VERITY 1910 MS Detector also features an off-axis vac-chip flange that reduces maintenance downtime and a new, all-metal vac-chip design that extends its lifetime. The detector uses less solvent, nitrogen, and power, lowering operating costs and providing a greener solution for mass detection,” Naza Lahoutifard-Henry, Director Portfolio Management-HPLC Business Line, added. The affordable VERITY 1910 MS Detector comes with all required accessories including the MiDas™ module, which consists of an integrated make-up pump, splitter, and direct sampling valve to deliver consistent flows to the VERITY 1910 Detector. The detector can be used with Gilson’s TRILUTION® LC software-controlled VERITY HPLC Systems and the PLC Purification Systems.

GLOBAL PHARMA | 38

Feature Note

VIP Ultra Low Freezers: Compressor Reliability Explained Managing Heat: How PHCbi Increases Reliability of Ultra-Low Temperature Compressors The Challenge How It Works To reach ultra low temperatures within a freezer it is often necessary to employ a cascade cooling methodwhereby two refrigeration systems work together to reach the desired temperature. The low-stage (B) removes heat from the freezer chamber while the high-stage (A) removes excess heat from the low stage system, which is ultimately released into the ambient air. The most important component of any mechanical refrigeration system is the compressor, which moves refrigerant through the system. Often compressors used in ultra low freezers are not purpose designed and have to operate beyond their intended limits causing increased temperatures and additional stresson the system â&#x20AC;&#x201C; a primary reason for freezer failure.

Function The PHCbi Solution ULT freezers employ application specific compressors that have been purpose designed for ultra-low temperatures. The specialist design reduces discharge temperatures and internal heat within the compressor. An oil cooling pipe with high-stage refrigerant loops around thecompressor reservoir (C) of both stages to cool the lubricating oil inside the compressor. While the oil lubricates internal bearings, the motor shaft (D) pumps an additional cooling spray directly to the discharge head (E) These innovative refrigerant feedback processesreduce compressor temperatures.

Performance

High stage refrigerant gas

High stage refrigerant liquid

Low stage refrigerant gas

Low stage refrigerant liquid

Using the Same Energy Twice High internal pressures and temperatures can have a number of detrimental effects o n ultra-low compressors. The specialist features of compressors reduce internal operating temperatures and therefore combat these effects to increase compressor lifetimes. Lubricant oil within the compressor ensures the continued smooth operation of the moving parts. This oil can become viscous or breakdown as a result of higherthan normal temperatures, resulting in increased wear of components, so it is vital to maintain its properties.

Enlarged Illustration of Compressor Sensors (not shown) Temperature sensors throughout the high and low stage circuits transmit information to the Panasonic controller for operation, monitoring and diagnostics.

PHCbi ultra low freezers are tested at high ambient temperatures to ensure optimum performance of the compressor even under demanding laboratory conditions.

Reliability Dependable Outcomes The specialist design of the compressors in the VIP ULT freezers ensures reduced temperatures and stresses within refrigeration systems for uncompromised performance and unsurpassed durability. When maintaining the integrity of precious samples is vital, VIP ULT Freezers offer the highest levels of reliability for storage yo can trust.

For more information, please visit our website: www.vip-ult-freezers.com

Nijverheidsweg 120 | 4879 AZ Etten-Leur | Netherlands T: +31 (0) 76 543 3839 | F: +31 (0) 76 541 3732 biomedical.nl@eu.phchd.com

www.phchd.com/eu/biomedical

GLOBAL PHARMA | 39

Feature Note

VIP Ultra Low Freezers: Compressor Reliability Explained Managing Heat: How PHCbi Increases Reliability of Ultra-Low Temperature Compressors

How it works A refrigerant is a fluid that transfers heat from one area to another. In a freezer, the refrigerant is used to cool the cabinet to the desired temperature by transferring heat from inside the freezer to the external environment through a cycle of thermodynamic processes. In a typical refrigeration system, the refrigerant gas is compressed, then cooled and condensed into a liquid. This liquid refrigerant then travels through a small capillary which increases the pressure of the refrigerant. When the high pressure liquid leaves the capillary, it enters a low pressure environment. This sudden change in pressure causes the liquid refrigerant to spray out of the capillary and evaporate. To revert back to its natural, gaseous state the refrigerant will need to absorb heat from its surroundings. It is this process of vaporization that draws heat from the freezer cabinet and this cycle will continue until the desired temperature is reached. The most important property of a refrigerant is its boiling point. This should be low in order to draw heat from the freezer to achieve the required temperature, but not too low as this presents problems with the condensing stage of the refrigeration cycle.The ideal refrigerant would have a boiling

point just below the desired temperature of the freezer. VIP ECO Ultra-low freezers use two hydrocarbon refrigerants. The low-stage refrigerant is used to remove heat from the freezer cabinet to provide a secure ultra-low (-86ยบC) storage environment. Since the lowstage refrigerant gas cannot condense back to a liquid at room temperature a second, high-stage refrigeration, circuit is used to cool a cascade condenser to around -40ยบC. Which is low enough to condense the refrigerant gas in the low-stage.

Ultra-low freezers, R-290 (propane) is used to remove the heat from the low-stage circuit. Propane has a boiling point of -42.1ยบC so provides an ideal temperature to condense the lowstage refrigerant. The low-stage refrigerant circuit uses R-170 (ethane) to remove the heat from the freezer cabinet itself. Ethane has a boiling point of -88.2ยบC which enables it to cool the freezer to ultra-low temperatures.

This system is known as cascade cooling. In the high-stage refrigeration circuit of High stage refrigerant gas

High stage refrigerant liquid

Low stage refrigerant gas

Low stage refrigerant liquid

PHCbi cascade cooling MDF-U76V VIP ECO ULT Freezer

Sensors (not shown) Temperature sensors throughout the high and low stage circuits transmit information to the Panasonic controller for operation, monitoring and diagnostics.

GLOBAL PHARMA | 40

Feature Note

VIP Ultra Low Freezers: Compressor Reliability Explained Managing Heat: How PHCbi Increases Reliability of Ultra-Low Temperature Compressors

How it saves: the benefits of hydrocarbon refrigerants Reduced running costs The use of highly efficient hydrocarbon refrigerants results in reduced energy consumption and therefore lower running costs. With key equipment and instrumentation operating continuously laboratories are able to significantly reduce running costs by investing in energy efficient facilities. PHCbi designs and builds advanced preservation systems to deliver maximum cost efficiency while maintaining the reliability and performance necessary for reliable storage of valuable research and clinical samples. Alongside a host of other energy saving features, the efficient cooling delivered by hydrocarbon refrigerants can allow smaller compressors to be used leading to significant reductions in energy use and running costs. In addition, more efficient refrigeration systems dissipate less heat to their surroundings reducing the requirement for air conditioning which can lead to further savings.

Increased efficiency The efficiency of a freezer can be measured as a coefficient of performance (COP) which is a ratio of the cooling provided to the electrical energy consumed. VIP point just below the desired temperature of the freezer.

VIP ECO Ultra-low freezers use two hydrocarbon refrigerants. The low-stage refrigerant is used to remove heat from the freezer cabinet to provide a secure ultra-low (-86ÂşC) storage environment. Since the lowstage refrigerant gas cannot condense back to a liquid at room temperature a second, high-stage refrigeration, circuit is used to cool a cascade condenser to around -40ÂşC. Which is low enough to condense the refrigerant gas in the low-stage. This system is known as cascade cooling. In the high-stage refrigeration circuit of ECO freezers have an extremely high COP. The efficiency of the ECO freezers is a result of the high latent heat of evaporation of the hydrocarbon refrigerants. This value relates to the amount of heat required to turn a liquid into a gas â&#x20AC;&#x201C; essentially the energy needed to break the forces which bind the refrigerant molecules together in a liquid state. As a larger amount of heat energy is required to break the binding forces between hydrocarbon molecules, these refrigerants

short atmospheric lifetimes and extremely low global warming potentials. The atmospheric lifetime of a gas relates to the average time that a molecule resides in the atmosphere before it is removed by chemical reaction or deposition. A short atmospheric lifetime means that the refrigerant is removed from the atmosphere quickly, resulting in a lower global warming potential and less impact on the environment. Refrigerants have a value for global warming potential (GWP) which relates to the warming effect of the gas in comparison with one unit of carbon dioxide. Propane and ethane have global warming potentials of 3.3 and 5.5 respectively. These values are significantly lower than traditional refrigerants.

remove heat more efficiently from the freezer cabinet than if a traditional refrigerant was used.

Extremely low environmental impact As well as being non-ozone depleting, hydrocarbons have

For more information, please visit our website: www.phchd.com/eu/biomedical

GLOBAL PHARMA | 41

Life Sciences

GLOBAL PHARMA | 42

Online Auction | April 18, 2019 Lab, Analytical and Bioprocessing Equipment from Teva Featuring HPLCs, Analyzers, Homogenizers, Lab Centrifuges, Freezers, Lab Mixers, Measuring Equipment and more from leading manufacturers such as Waters, Sotax, GEA and Eppendorf.

Auction Details: Bidding Starts: April 18, 2019 - 9:00 U.K. Time Lots Begin Closing: April 18, 2019 - 16:00 U.K. Time Location: Bridgend, UK & Haarlem, Net (Europe) www.EquipNet.com/auctions

Please contact EquipNet auctions with any questions or to schedule an inspection: auctions@equipnet.com | +44.0.118.901.6161 | www.EquipNet.com/Auctions

EquipNet is a global leader of surplus asset management solutions. Weâ&#x20AC;&#x2122;re recognized for our proprietary asset management platform, our revolutionary industrial equipment marketplace, and our results-driven project management services. Our clients span across multiple industries and range in size from small businesses to Fortune 500 multi-national corporations and leading regional manufacturers. EquipNet solutions deliver maximized financial return while improving our clientâ&#x20AC;&#x2122;s corporate image, adhering to their safety standards, and contributing to their sustainability initiatives.

GLOBAL PHARMA | 44

Started just outside Boston, Massachusetts in 1999, we have grown to having offices across 25 locations worldwide. Our associates are customer focused and want to help you find the right equipment to meet your needs, as well as offering the best solution for selling your surplus equipment. Our services include: Appraisals, Asset Redeployment Management System, Auctions, Worldwide Logistics, Site Closure Management, and much more. Visit our website at equipnet.com to find out more about EquipNet and our services.

EquipNet is actively engaged with over 377,000 qualified buyers from all over the world. Our international team speaks over a dozen languages making communication with our clients easy and seamless. We conduct business in more than 170 countries and we serve more than 20 different industries. No matter what you are looking for, you can rest assured that you will find it on our MarketPlace across the 10,000 active listings at any given time. This year, EquipNet is celebrating 20 years in business. The company has a focus on pharmaceutical, biotech, chemical, food & beverage, personal care, as well as electronics and industrial manufacturing markets. EquipNet offers a broad range of services for the effective tracking, redeployment, sales or purchasing of second-hand capital assets, helping its

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clients generate a significant amount of previously unidentified funds from laboratory instrumentation and manufacturing equipment that have been sitting idle. Taken individually, the company’s services help customers meet challenges such as tracking and redeploying assets within a company’s enterprise that it can no longer use, needing to buy equipment quickly and economically, and moving machinery around the globe. When combined, these and other EquipNet services provide a comprehensive and seamless asset management program to maximize investment recovery. Roger Gallo, President and CEO said, “It’s a great milestone for EquipNet. It’s gratifying to see the positive financial impact that EquipNet has made on to our clients’ bottom line

European Headquarters EquipNet Europe Unit 3, The Pavilions, Ruscombe Business Park Twyford Berkshire RG10 9NN, UK Tel: +44.0.118.901.6161, Fax: +44.118.901.6162 eusales@equipnet.com

while significantly contributing to landfill avoidance efforts on a global scale.” Twenty years on, EquipNet has grown from two people in a crowded back room of borrowed space, to now more than 150 people in over 20 locations through North America, Latin America, Europe and Asia. EquipNet provides full asset management programs to some of the biggest corporations in the world, including Novartis, Unilever, Teva, Mars Wrigley, Tesla, and many others. To find out more about how EquipNet can help you with your investment recovery and sustainability efforts, visit our brand new website at www.EquipNet.com.

North American Headquarters EquipNet, Inc. 5 Dan Road Canton, MA 02021 Tel: +1.781.821.3482 Fax: +1.617.671.1269

GLOBAL PHARMA | 45

Chromotography special feature

GLOBAL PHARMA | 46

Antibody Capture and Analysis

TOSOH RESINS AND COLUMNS SPECIFICALLY OPTIMIZED TO MEET YOUR NEEDS Tosoh purification resins and analysis columns have a long set standard in industrial manufacturing of biotherapeutics. Sustained investment in research and development maintains our position at the technological frontier. Over the past decades, we have brought to market successive generations of resins and columns that anticipate our clientsâ&#x20AC;&#x2122; evolving needs in response to growing pressures on time-to-market, needs for control of production price, tightened safety controls and increasing titers.

PROTEIN A HC LEACHING REMAINS UNDER THE 10 NG/ML LIMIT IN ALL TESTING CONDITIONS 10

HIGH STABILITY TO ENSURE A LOW DEGREE OF PROTEIN A LEACHING FOR GREATER SAFETY

Limit of 10 ng/mL

Whatever the titer (feed concentration) Whatever the pH

Whatever the load

Regardless of the initial load and pH, Protein A leaching is typically kept well beneath the limit of 10 ng/mL, guaranteeing high purity and safety; the leaching even diminishes slightly as the titers are increased. This low leakage is due to the ligands’ multipoint attachment to the TOYOPEARL® polymer matrix.

Protein A leaching

Tosoh Protein A HC* chromatography:

Figure 1

An optimized combination of high stability and high binding capacity

The Protein A HC resin’s chemical and thermal stability allows at least 300 Cleaning-in-Place (CIP) cycles (at 0,2 M NaOH) without significant reduction in dynamic binding capacity (DBC). The caustic stability during cleaning, due to its multipoint attachment, considerably reduces operating costs.

NO SIGNIFICANT REDUCTION IN DBC AFTER 300 CIP CYCLES

DBC at 10% breakthrough (mg/mL)

HIGH ALKALINE STABILITY TO PERMIT MULTIPLE REUSE AND THEREFORE REDUCED COSTS

80 60 40 20 0

100

150

200

250

300

Number of CIP cycles

Figure 2

*TOYOPEARL AF-rProtein A HC-650F

The TOYOPEARL Protein A HC resin displays a binding capacity of > 68 g IgG/L. This exceeds the typical binding capacity of 30-50 g/L on competitor Protein A resins. Since the share in the overall cost of purification accounted for by Protein A capturing diminishes as resin capacity increases (due to even smaller in-process pools, reduced buffer consumption, etc.) this reduces total operating costs substantially.

THE COST OF PURIFICATION STEP DROPS FROM 43% TO 21% Protein A purification step as a % of operating costs

HIGH BINDING CAPACITY TO REDUCE THE COST OF PURIFICATION

50% 45%

43%

40%

Typical binding capacity of Protein A HC

35% 30% 25% 21% 20%

Resin binding capacity (g/L)

Figure 3

IgG DBC (g/L)

MORE THAN 50 g IgG/L IS ALREADY CAPTURED AFTER ONLY 2 MIN

80 70 60 50 40 30 20 10 0

IgG @ 1 mg/mL IgG @ 5 mg/mL IgG @ 10 mg/mL

HIGH BINDING CAPACITY EVEN AT VERY SHORT RESIDENCE TIMES TO REDUCE PROCESS TIME In contrast to many Protein A resins on the market, the binding capacity of the Protein A HC resin is high even at residence times as short as two minutes, reducing total process time.

3.3

Residence time (minutes)

Figure 4

HIGH BINDING CAPACITY TO MEET INCREASING UPSTREAM PROCESSING LOADS AND REDUCE COSTS

LESS RESIN IS NEEDED TO TREAT THE SAME AMOUNT OF FEEDSTOCK

The rising titers that result from improvements in upstream efficiency are a challenge for downstream processing (DSP), especially at the Protein A-capturing step. The protein adsorption capacity of the Protein A HC resin prevents DSP bottlenecks at the highest possible titers (10 g/L), even at short residence times. A Protein A HC resin capacity of even more than 100 g/L has been achieved. This exceeds the dynamic binding capacity of all other commercially available alkaline-stable Protein A resins. This outstanding characteristics reduces both the volume of resin needed and the footprint, which minimizes the costs. Figure 5

TSKgel® SW SERIES, THE GOLD STANDARD FOR QUALITY CONTROL ANALYSIS OF ANTIBODY THERAPEUTICS

Tosoh columns for quality control of mAbs by SEC:

For decades, TSKgel G3000SWXL columns have been the industry’s standard for antibody analysis and quality control of monoclonal antibodies by size exclusion chromatography (SEC). We have developed new series of silicabased SEC columns for HPLC and UHPLC providing shorter analysis time and higher resolution. Their principal characteristics are: • • • •

Higher resolution with SuperSW mAb HR Faster separation with SuperSW mAb HTP Higher molecular weight range with UltraSW Aggregate Highest resolution and fastest separation with UP- SW3000 UHPLC columns

a range tailored to meet your needs

TSKgel UP-SW3000 OFFER HIGHER RESOLUTION ACROSS THE WHOLE MOLECULAR RANGE RS (peak 2/3): 3,56 vs 3,47

2 RS (peak 1/2): 1,52 vs 1,25

Tosoh TSKgel columns are designed to improve resolution of antibody fragments, monomers and dimers. The SuperSW mAb HR, the TSKgel UltraSW Aggregate and the TSKgel UP-SW3000 column offer superior resolution over the whole molecular weight range, from fragments to aggregates.

Tosoh TSKgel

1 4 1

Competitor

3 4 5 Retention time (min)

ANALYSIS TIME TWICE AS FAST

TSKgel G3000SWXL

6 8 10 Retention time (min)

Figure 7

LOT-TO-LOT REPRODUCIBILITY TSKgel SW SEC columns are known for their outstanding quality and reproducibility. The new series columns are highly reproducible for HPLC as well as for UHPLC systems. This allows straight forward method validation and routine implementation.

The new series of Tosoh columns were also engineered to provide shorter analysis time by: • Increasing the throughput without compromising resolution with SuperSW mAb HTP (HTP for “High ThroughPut”) • Completing the analysis in half the usual run time and increasing resolution with the use of the UP- SW3000 column on a UHPLC system. HIGH LOT-TO-LOT REPRODUCIBILITY

UV absorbance @ 280 nm (mAU)

Detector response (mAU)

UP-SW3000 columns

Figure 6

BETTER RESOLUTION IN HALF THE TIME

UV 280 nm

AN ALWAYS HIGHER RESOLUTION

Lot Lot B Lot C

Lot D 3

5 7 9 11 Retention time (min) - TSKgel UP-SW3000

NEW! - BIOPROCESSING APPLICATION NOTEBOOK Discover the best methods for the purification of biomolecules! Request a copy of the application notebook now at bit.ly/BioProcessing

ANALYSIS

APPLICATION NOTE

CORRELATION OF FcR AFFINITY CHROMATOGRAPHY WITH GLYCAN PATTERN AND ADCC ACTIVITY OF A THERAPEUTIC ANTIBODY

Fc RECEPTOR AND ADCC ACTIVITY

Glycoforms of an antibody sample can be partly separated based on the strength of their binding to the FcR ligand. Resulting peaks can be assigned to low, medium, and high ADCC activity (Figure 2). FCγR -AFFINITY CHROMATOGRAPHY

Mid activity

ABS 280nm

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action (MoA) of monoclonal antibodies used in cancer treatment. Selecting suitable cell lines and optimizing culture conditions towards expression of antibody candidates with desired ADCC activity is an essential part of the R&D process. A fast and straight forward approach to easily access ADCC activity would facilitate screening of a large number of clones or monitoring the effect of upstream process variations. Other stages of R&D and production could benefit from fast ADCC assessment as well: comparing biosimilar and originator, detecting lot-to-lot variations, monitoring product stability, to name but a few.

Low activity

High activity

10 15 Retention time (minutes)

Figure 2

ADCC starts with the binding of the Fab region of an antibody to a target cell, e.g. a cancer cell. Binding of the Fc domain of that antibody to Fcγ receptors on the outer membrane of natural killer (NK) cells triggers degranulation into a lytic synapse and finally the apoptosis of the cancer cell (Figure 1). The glycan micro-heterogeneity of the Fc domain, in particular on the galactose and core-fucose levels1), influences binding of the Fc domain to Fcγ receptors.

Taking the well-known therapeutic antibody rituximab as an example, this application note demonstrates that the pattern of FcγRIIIa affinity chromatography shows a good correlation with the results obtained by ADCC reporter assay. Fractions collected from HPLC peaks with different receptor affinity also show different glycosylation patterns at the Fc domain. Mid affinity

ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY

RITUXIMAB

Current ADCC activity tests are either cell based bioassays or surface plasmon resonance (SPR) measurements using immobilized Fcγ receptor. A new approach combines the specificity of the FcγIIIa receptor (FcγRIIIa) with the easy handling of an HPLC method.

Natural killer (NK) Antibodies Signaling through cell FcγRIIIa bind antigens FcγRIIIa triggers the on the surface receptors recognize release of cytokines cell-bound of target cells and cytotoxic granuals antibodies that inﬁltrate the target cell

ABS 280nm

For Fc receptor affinity chromatography, a recombinant FcγIIIa receptor ligand is immobilized on a stationary phase.

Rituximab (Figure 3) is a recombinant chimeric human/ mouse monoclonal IgG1 antibody, approved in 1997 and used to treat certain autoimmune diseases and types of cancer. Besides other effects of rituximab, its Fc portion mediates antibody-dependent cellular cytotoxicity (ADCC) 0 5 10 15 20 Retention time 2) (minutes) and complement-dependent cytotoxicity (CDC) . N-glycans bound to the Fc domain of Rituximab contain mainly G0F and G1F structures.

Tumor cells die by apoptosis

Original image by Satchmo2000, distributed under a CC-BY 3.0 license.

Figure 1

Figure 3

Low affinity

High affinity

ANALYSIS

FcR AFFINITY CHROMATOGRAPHY OF RITUXIMAB In FcγRIIIa affinity chromatography, purified antibody or cell culture supernatant is injected under conditions that promote binding of mAbs to the FcγRIIIa ligand. Elution of bound mAb variants is performed by lowering the pH of the mobile phase in order to disrupt the target/ligand interactions. The higher the affinity of a mAb variant to the receptor, the higher the retention time of the respective peak. FcR affinity chromatography analysis of rituximab on the new TSKgel FcR-IIIa-NPR column results in three peaks representing variants with low, medium, and high FcγRIIIa affinity (Figure 4a). For subsequent characterization of the three peaks a semi-preparative prototype FcR-IIIA column was used to collect fractions.

ADCC BIOASSAY OF RITUXIMAB AND FcR AFFINITY FRACTIONS ADCC activities of the original rituximab sample and the three fractions collected from semi-preparative FcR affinity chromatography were determined with the ADCC reporter bioassays (Promega). The Fc effector reporter bioassay uses the FcγR and NFAT-mediated activation of luciferase activity in effector cells to determine ADCC efficacy and potency of antibodies. Figure 5 shows the ADCC reporter bioassay response to rituximab and to the three fractions collected from FcR affinity chromatography (low, medium, high FcγR affinity). ADCC REPORTER BIOASSAY RESPONSE TO RITUXIMAB

Figure 4b shows that the separation pattern on the semipreparative column is identical to that of the analytical column, although resolution is worse. Fractions out of the three peaks were collected. For each peak, cleaved and 2AB-labeled N-glycans were characterized by HILIC-UHPLC and ADCC activity was analyzed with a standard ADCC reporter bioassay kit (Promega). FCγR AFFINITY ANALYSIS OF RITUXIMAB ON TSKgel FCR-IIIA-NPR (A) AND A PROTOTYPE SEMI-PREPARATIVE COLUMN (B)3)

Figure 5 ADCC reporter bioassay response to rituximab (grey), fraction 1 (low FcRγ affinity, blue), fraction 2 (medium FcγR affinity, red) and fraction 3 (high FcγR affinity, green)3) EC 50 VALUES OBTAINED BY THE REPORTER BIOASSAY TEST Antibody

EC 50 (µg/mL)

Rituximab

0.098

Peak 1

0.153

Peak 2

0.072

Peak 3

0.049

Table 1 Table 1 shows the EC50 values obtained by the reporter bioassay test. The lower the EC50 value, the higher the ADCC potency. As expected, peak three (high FcRγ affinity, green) shows highest ADCC potency in the bioassay. Peak two shows intermediate, and peak 1 shows lowest ADCC efficacy and potency. ADCC efficacy and potency of the original rituximab lies between the low and medium affinity fractions.

Figure 4

ANALYSIS

GLYCAN ANALYSIS OF FCR AFFINITY FRACTIONS

CONCLUSION

The glycan profile of the collected fractions was analyzed by hydrophilic interaction chromatography. Figure 6 shows the glycan pattern of the FcR affinity fractions compared to a glycan library. The antibody glycoforms collected in peak 3 (highest affinity) show mainly galactose containing N-glycans (G1F and G2F). Peak 2 glycoforms contain more G0F glycans than peak 3 and glycoforms collected in Peak 1 (lowest affinity) show predominantly fucosylated glycans without galactose units (G0F).

The ADCC activity bioassay results show that high retention on TSKgel FcR-IIIA-NPR corresponds to a high ADCC activity. The HILIC-UHPLC glycosylation pattern analysis of the FcR affinity fractions also matches the common understanding that terminal galactose units of Fc-glycans typically enhance affinity to FcγRIIIa and ADCC activity while core fucose units decrease ADCC activity of antibodies. These results confirm that FcγRIIIa affinity chromatography allows fast assessment of biologic activity and glycoform pattern of antibodies.

HILIC ANALYSIS OF OLIGOSACCHARIDES OF THE THREE FcR AFFINITY FRACTIONS

REFERENCES: 1) 2) 3)

D. Reusch and M. L. Tejada, Glycobiology 2015, 25 (12): 1325-1324; doi: 10.1093/glycob/cwv065 G.J. Weiner; Semin Hematol. 2010; 47 (2): 115-123; doi: 10.1053/j. seminhematol.2010.01.011 Master Thesis Leila Ghaleh, TU Darmstadt

TSKgel is as registered trademark of Tosoh corporation. Rituximab kindly provided by Rentschler Biopharma

Figure 6 HILIC analysis of oligosaccharides of the three FcR affinity fractions (Peak 1 blue, Peak 2 red, Peak 3 green) compared with a 2-AB labelled biantennary glycan library (grey) 3)

A brief overview of readily available Liquid Chromatography Techniques

Ade Kujore, March 2019

Nowadays the term liquid chroma-

tography (LC) covers so many different techniques. The one attribute which these techniques share is that that components or analytes within liquid samples are separated by virtue of a liquid (mobile phase) passing through a solid material (stationary phase). Components are detected by some form of a detector.

Each technique may be used to detect, characterise and/or quantify specific analytes within many sample matrices. Liquid chromatography techniques include the following. Paper Chromatography is probably the simplest form of liquid chromatography, where a high grade porous paper is used in combination with liquid/s, to obtain semi-quantitative and semi-qualitative determinations of a wide range of analytes. The method of detection is normally visual sometimes in combination with a visualisation chemical such as ninhydrin. Paper chromatography can be up scaled on a semi-preparative basis to produce quantities of

crude analytes. Thin Layer Chromatography (TLC) is similar to paper chromatography, except that instead of paper, solid particles are coated onto to plates of glass or inert plastics. A development of TLC is HPTLC or High Performance Thin Layer Chromatography. Here the solid particles used are much finer than those of TLC, so that higher resolution separations may be effected and quantitative results may be obtained. HPTLC can be automated, so can be useful for high throughput screening and for semi-preparative separations. Capillary Electrophoresis (CE) is an analytical technique with several variants, DNA fragments, glycoproteins, nucleic acids and ions may be separated in complex matrices, due to their electrophoretic mobility after a voltage is applied to the analytes. Highly sensitive qualitative determinations of complex samples, such as dye mixtures in the presence of blood splatters are possible. Low Pressure Column Chromatography is another simple form of liquid

chromatography, where the liquid is passed through a usually long glass column packed with a solid particles. Aliquots of eluates are often analysed offline. The technique can be useful for some easily denatured analytes. Up scaling on a semi-preparative or preparative basis is possible. Fast Protein Liquid Chromatography is a form of medium-pressure chromatography similar in principle to low pressure column chromatography. But where the liquid is pumped through a usually glass or plastic column packed with a solid particles and detection can be online, through the use of UV/Visible, pH, conductivity or other detectors. High Performance Liquid Chromatography (HPLC) is a form of liquid chromatography, where liquid/s is pumped at high pressures (to around 40 MPa) through a usually stainless steel or PEEK column, packed with a solid particles. The resulting eluate is passed through an online detector. The technique is commonly used for quantitative and qualitative analysis of an enormous range of analytes.

GLOBAL PHARMA | 55

There is a myriad of column types and possible liquids, these combinations will effect different separations, depending on the sample matrix and analyte. Different detectors are used, depending on the type of analytes to be detected. Up scaling on a semi-preparative or preparative basis is possible. Ion Chromatography (IC) is a variant of HPLC, where anion analytes, such related organic acids, groups of transition metals, or groups of cations, may separated. Detection is normally by use of an online conductivity detector and sometimes UV/Visible and electrochemical detectors. Quantitative and qualitative determinations are possible. IC can be up scaled on a preparative and semi-preparative basis to produce quantities of the required pure analytes. Size-exclusion chromatography (SEC) is an HPLC technique where analytes are separated and detected by virtue of their molecular weight. It is most often used in quantitative and qualitative determinations of large molecular weight analytes, such as antibodies, haemoglobins and large organic polymers such as polystyrenes and plastics. The most common types of detectors used here are refractive index, light scattering and infra red. SEC can be up scaled on a preparative and semi-preparative basis to produce quantities of the required pure analytes.

and used for high throughput screening. Mass Spectrometry (MS) can include a range of liquid chromatography analytical techniques, where after passing through an HPLC or a capillary electrophoresis system, analyte/s are ionised, then passed through a mass analyser and the resulting mass-to-charge ratio ion current is detected. Many types of mass spectrometry of varying complexities are available. A wide range of analytes, especially those within a group, may be detected and characterised quantitatively and qualitatively. In addition, mass spectrometry can elucidate the structure of molecules within complex samples, hence is used extensively in pharmaceutical and agrochemical development, metabolomics, proteomics, forensics, clinical screening, isotope ratioing and protein sequencing. Each LC technique has its own inherent degree of sensitivity and specificity for any appropriate analyte. The type of stationary phase, mobile phase, detector choices and settings, also play an effect. In addition within each technique, analyte sensitivity is influenced by factors such as interferences from sample matrix, sample pre-treatment such as extraction and pre-concentration.

technique where two or more LC detectors are used in any given analysis. HPLC, SEC, FPLC and IC sometimes use two or more detectors in series or parallel. The use of detectors in series or parallel as in 2D and multi-dimensional hyphenated systems helps to shorten the analysis time of a single sample. Different types of analyte groups within the same sample, may be analysed with high specificity and sensitivity, from the insertion of one single sample, with two or more different mobile phases, stationary phases, flow paths and detectors. The initial capital cost of LC techniques varies wildly, with paper chromatography costing just a few pence to a million or so pounds for Fourier Transform mass spectrometry instruments. An analyst must decide on why they need the instrumentation, taking into account the numbers and types of samples involved, potential changes of use over time, computing requirements and servicing and maintenance costs. Despite most liquid chromatography techniques and instrumentation being mature, the concepts are continuously being enhanced. This is partly fuelled by the need to reduce the use of organic solvents and other materials, decrease analysis and sample preparation times, increased simplification of operation, wider ranges of applications and space saving designs.

Mass spectrometry is not the only LC

UHPLC, Ultra High Performance Liquid Chromatography is the same as HPLC, but pump pressures of up to 100 MPa are used with finer sized column particles, to effect faster and possibly higher resolution separations than with HPLC. Supercritical fluid chromatography (SFC) is a form of HPLC in which a supercritical liquid such as pressurised carbon dioxide, is pumped through a column packed with solid particles. It may be used for the analysis of low to medium molecular weight, chiral analytes. Nano-flow chip integrated circuits (Lab-on-a-Chip) are miniaturised devices, where minute amounts of liquids may be passed through solid particles. They may be automated GLOBAL PHARMA | 56

Laboratory identification solutions from Brady Precise, reliable and efficient. Just like you! Brady’s laboratory identification products are designed to serve the world’s biotechnology, agricultural, environmental and forensic researchers. Whether you work in pathology, histology, chromatography or other areas of science, Brady labelling solutions can help you maintain Good Laboratory Practice.Discover our durable sample identification labels and the systems you can use to print them in our Sample Identification Guide Book

‘Never lose a sample again’. BRADY Corporation Tel: +44 (0) 1295 228 288 emea_request@bradycorp.com www.bradyeurope.com/lab GLOBAL PHARMA | 57

Index

GEA Group

Adamus SA

Natoli Engineering Company

Source BioScience

BENEO GmbH

Molecular Devices

Priorclave Ltd

BioGenes GmbH

Gilson Company Inc

PHC Europe B.V

EquipNet Inc

TOSOH Chemicals company

Ade Kujore

BRADY Corporation