Biologics Reshaping Pharmaceutical Market

BIOLOGICS

RESHAPING PHARMACEUTICAL MARKET The Biotherapeutics market has gained substantial attention and evolved as a profit-making segment of the pharma industry for a decade. This article sheds light on how next-generation proteins are reshaping the market and talk about innovative strategies that pharma companies are thinking of to cut down the developmental cost incurred for producing a safe and efficacious biotherapeutic product and improvising patient experience.

Prasanthi Polamreddy, Scientific Manager Excelra Knowledge Solutions Pvt Ltd.

The biotherapeutics market is booming. Currently, annual growth rate of biologics is double that of conventional pharma and is forecasted to grow rapidly. Increase in incidence of chronic diseases like cancer, diabetes, autoimmune diseases, genetic conditions…etc and acceptance of biotherapeutics because of their high efficacy and safety are some of the major factors driving the biopharmaceutical market. Also, biologics gained edge in diseases that could not be successfully targeted by small molecule drugs especially oncology indications and autoimmune ailments. And today biotherapeutics industry moved one more step ahead and invested in the development of next generation biologics which forms the current era. However, despite the considerable demand, high cost associated with biotherapeutics restricts market growth.

Evolution of biotherapeutics

Since the launch of breakthrough therapeutic drug – Insulin (enzyme replacement therapy) in 1982, the growth potential of biologics has been promising. In its early days, this industry faced some serious concerns. Despite vast challenges in its path, this industry evolved as multibilliondollar market and today 7 of the top 10 bestselling drugs are biologics. Many innovative therapies were launched till date and comprise vaccines, enzymes-based therapies, cell therapies, gene therapies, immunotherapies, peptides and protein products. Monoclonal antibodies (mAbs) revolutionised the biotherapeutics market and rose to leading position among the other biologic products.

Antibody drug conjugates

The increased scale of demand for effective biologics, especially monoclonal antibodies and continuous efforts to improvise the therapeutic potential led to advancement of protein engineering technologies which further fuelled the development of next generation biologics. These next generation biologics are characterised by longer half-life and low immunogenicity and comprise antibody drug conjugates (ADCs), bi-specific and multi-specific antibodies, fusion proteins, engineered cells and antibody-like proteins (ALPs). ADCs are one of the lucrative classes of engineered proteins and were proven to be effective in combating cancer, especially haematological malignancies and breast cancer. Though the side effect profile of ADCs is comparable to that of chemotherapeutics, it has not dampened their development and rather intensified ADC R&D. Currently there are almost 600 clinical trials focused on ADCs of which 40 trials are evaluating ADC and check point inhibitor combinations in oncology indications.

Fusion Proteins

Another promising class of next generation biologics comprises therapeutic fusion proteins. Fusion protein product is obtained by genetically fusing two protein domains encoded by two different genes and hence exhibits multiple functional properties. There exist several categories of fusion proteins depending on the type of proteins that were fused. One of the predominant types among these fusion proteins is the Fc-fusion protein where Fc fragment of IgG is fused with protein or peptide. And the first fusion protein to get FDA approval is the Fc-fusion protein (Etanercept (Enbrel) in 1998). Since then many Fc-fusion proteins have been approved in the major markets of USA, Europe and Japan and include Abatacept (Orencia), Belapacept (Nulojix), Aflibercept (Eylea), Rilonacept (Arcalyst), Romiplostim (Nplate), Dulaglutide (Trulicity), Albiglutide, Efmoroctocog alfa (Elocta), Asfotase alfa (Strensiq). Apart from Fc-fusion proteins, some of the other types of fusion proteins (where a protein has been linked to either peptide/ kinase/toxin/cytokine/antibody fragments/ albumin/transferrin/blood factors) have been successful in the market. Given the commercial success as evidenced by many approved therapies of this class, it is very likely that fusion proteins would emerge as promising therapies in the years to come.

Bispecific antibodies

Fusion proteins were also designed to interact with two or multiple target proteins and exhibiting bi- and multifunctional characteristics. Bispecific antibodies (bsAbs) hold great promise as therapy and swiftly expanding with more potential for commercial success. Targeting two antigens at a time with bsAbs was not a very new concept and was highlighted with the approval of catumaxomab. In Spite of considerable side effects associated with this therapy, the spark was retained and created surge in R&D and investment and these continuous efforts resulted in the approval of two therapies- blinatumomab (binds to CD3 on T cells and the CD19 antigen on tumour cells) and emicizumab (binds to the FIXa and FX zymogen). Currently, almost 85 bsAbs are in the clinical developmental pipeline and among these 86% are targeted for oncology indications (AMG 420, REGN1979 and XmAb14045) reflecting a considerable interest in their development as cancer therapy. Though a good number are in the early phase of clinical development pipeline these are yet to be proved as commercially viable.

Gene therapies

Gene therapies are another class of biologics witnessing success in the market and expected to grow tremendously in the near future. Despite the many obstacles and failures in the initial phases, gene therapies have seen huge progress in the recent past and are being used to target life threatening disease like cancers and nerve degenerative diseases. Since the

approval of first gene therapy in 1990 for ADA-SCID (Severe Combined Immunodeficiency due to Adenosine Deaminase deficiency), ~10 therapies did hit the global markets. Luxurna, developed by Spark Therapeutics is the first human gene therapy approved in US (in 2017) for Retinal dystrophy. In the same year, first CAR-T therapy developed by Novartis (KYMRIAH) received the approval and Kite Pharma’s YESCARTA was approved shortly thereafter. CAR-T therapies generated by engineering T-cells revolutionised the cell and gene therapy research and continue to receive a good amount of investment in the R&D perspective. These therapies marked a milestone in this new field and generated an enormous amount of interest among academia, research institutes and pharma companies. Currently a good number of CAR-T therapeutic leads are in mid- to late stage of clinical development and this field is poised for success soon. Next generation gene therapies based on CRISPR platform also hold great promise and poised to grow impact biotherapeutics market significantly. Currently, hundreds of therapies are in clinical development and this growing popularity can be correlated with considerable increase in capital investment to fund research activities.

Phage therapy, though sounding new is perhaps a century old concept. In phage therapy a bacteriophage will be used to kill bacteria and hold huge promise in tackling antibiotic resistance. Currently there are no approved therapies in healthcare, but seems things are moving in the direction of success and might become more common soon.

Challenges with biotherapeutics

However, biologics do have disadvantages. (a) Their inherent structural complexity increases the developmental time and cost (b) Their high molecular weight and complex structures impede them from crossing the cell membrane and targeting intracellular molecules (c) Complex mechanism of action (d) formulation challenges like aggregation and degra

Advancement of protein engineering technologies has steered in unprecedented opportunities to develop safe and efficacious biotherapeutics.

dation (e) Limited delivery mechanisms. Most of the biologics should be administered intravenously and this requires multiple hospital visits life-long in case of chronic conditions and is cost intensive (f) complex manufacturing and characterisation processes. As biologics are produced from live cells, selection of right cells and cell cultivation conditions hugely impact the production of the quality product with good yield. And characterisation of biologics is difficult compared to small molecules (g) cell expansion (required numbers) being bottleneck for cell therapies (h) clinical testing is associated with huge financial burden.

Innovations in the field

Biopharmaceutical companies are also thinking of ways to maximise the therapeutic window of biologics and harness novel manufacturing processes, formulation and delivery device technologies to ensure production of quality and stable product, to make the product more affordable and improvise the experience of patients. Genentech has developed a second generation anti-CD20 Ab (Gazyva) by glycol-engineering Fc region of original anti-CD20 Ab (Rituximab) to enhance Fc gamma receptor IIIA binding which is essential for cytotoxicity functionality. Gazyva was the first glycol-engineered antibody to get FDA approval and this drug was also granted break-through designation. Genentech’s Trastuzumab (Herceptin), in intravenous form had been approved in 1998 and is considered as breakthrough drug for treatment of HER2+ breast cancer. Later, with an objective to improve patients compliance, Genentech developed subcutaneous form of drug (Herceptin Hylecta) which received FDA approval in 2019. The safety of subcutaneous form of the drug is comparable to that of intravenous form. Takeda is also presently evaluating the efficacy and safety of a new subcutaneous formulation of monoclonal antibody vedolizumab (Entyvio) in adults with moderate to severely active Ulcerative colitis (UC) or Crohn's disease (CD) which is currently being administered intravenously. Formulating biologics into oral dosage forms will significantly reduce the delivery cost. But, whether gastric pH, gastric enzymes and epithelial cells lining the gastrointestinal tract would help the orally administer biologic product in achieving the required bioavailability is a critical factor to be considered. Because these challenges were observed with the oral form of insulin. This further led to the development of inhaled insulin therapy by Pfizer which was approved by FDA in 2006. Another innovation that comes in this direction is the RaniPill™capsule developed by Rani Therapeutics. Delivery of drugs through this robotic pill was equivalent to injections in pre-clinical and human trials are underway.

Other paths that researchers are looking for consideration are, using wearable drug delivery devices as a solution. FDA approval of Pushtronex™ system (on-body infusor with prefilled cartridge) for Repatha (Evolocumab), a hands-free device designed to provide 420 mg of Repatha in a single dose is another milestone in the course. But, will success of Pushtronex™ system boost the innovation in this angle? Given increased volume of data and increasing importance of artificial intelligence which is being embraced by all other industries, pharma companies which

thrive on innovation are also thinking of adopting this technology to decrease developmental costs and increase success rates. Leveraging artificial intelligence for predicting the right parameters and conditions required for bioprocessing and thereby bring down the developmental costs and producing high quality product is something which biotherapeutics industry is contemplating on. In context of cell therapies where obtainment of relevant number of cells with viability, potency and safety is currently challenging, use of analytical technologies might create a new wave in manufacturing of cell therapies. Artificial intelligence would also hold great promise in early drug discovery phase more predominantly target identification which is crucial for success rate of therapeutic products. Would artificial intelligence become magic bullet in biologics manufacturing? All biotherapeutics are produced from living cells, majorly CHO cells and hence sensitive to manufacturing processes. Giant biologic manufacturers

are looking for alternatives to mammalian cells so as to decrease the production cost and increase product quantity. Biogen in collaboration with Bill & Melinda Gates Foundation and MIT Center for Biomedical Innovation have engineered eight alternative hosts including fungal, algae, and trypanosome systems for the production of full-length mAbs.

Future vision

Finally, advancement of protein engineering technologies has steered in unprecedented opportunities to develop safe and efficacious biotherapeutics. Investment of huge efforts on optimising existing therapies to make drug targeting better, enhancing functionality and improvising patient experience through new formulation and drug delivery devices suggest prominent swift towards biotherapeutics market in the years to come. Also, changes in the FDA regulations (removed a rule under Section 610.21 of the FDA code) implemented recently pertaining to biologics would further boost the approval rates for biotherapeutic products. Hence, with this advancement in technology and increased investment on innovation, biotherapeutics products will continue to enrich medicinal armoury and make a foot print in the near future.

References are available at www.pharmafocusasia.com

AUTHOR BIO

Prasanthi Polamreddy is a pharma professional with over 10 years’ experience in standardizing and analyzing pharmacological data, computational drug discovery, and development of pharma competitive intelligence reports. She currently works with Excelra Knowledge Solutions Pvt Ltd as a scientific manager.

TISSUE ENGINEERING Choosing low-endotoxin gelatins for cell cultures

Barbara Vanhoecke, Innovation Manager Biomedical, Rousselot

Tissue engineering involves the creation of functional constructs for therapeutic purposes that restore or improve damaged tissues or whole organs in patients.

The process requires three essential components: an appropriate three-dimensional scaffold to support transplantation and growth of cells; cells, which can be reparative cells, that are able to form a functional matrix; and biologically active molecules, such as cytokines and growth factors that promote the formation of the desired tissue type.

One of the preferred methods to build the scaffold is with the use of hydrogels, polymer-based substances with a high water content that replicate

the characteristics of the extracellular matrix. As a natural, well-known ingredient, gelatin holds a huge potential in tissue engineering and gelatin-based hydrogels are a popular choice thanks to their biocompatibility and biodegradability.

In the following interview, Barbara Vanhoecke, Innovation Manager Biomedical at Rousselot, explores the value of low-endotoxin X-Pure gelatins already in early stage research and developments in field of tissue engineering, which might eventually go into the clinic with a strong safety profile.

1, What are the current challenges for tissue engineering?

Despite the significant investment in scientific research, the scalability and the undeniable potential, there are only a few current clinical applications of tissue engineering and even for those commercial success has been difficult to achieve. One of the primary challenges is the lack of a unified regulatory approach, as the fast-moving and complex nature of the industry outpaces the ability of regulatory frameworks to keep up.

But there are also a number of biological challenges. Before implantation the host cells are expanded and grown in tissue culture. Implanting scaffolds populated by these tissuegrown host cells is challenged by the generally low survival in vivo of the host cells. Another major challenge is revascularisation of implanted tissues/organs at the human scale. Moreover, there are major risks associated with tissue engineering due to potential tumorigenicity, immunogenicity and rejection of the graft. However, by using biomaterials that closely mimic the body’s own matrix and by avoiding unnecessary immune responses these risks can be greatly minimised. Further, it is critical that the biomaterial used to replicate the

Barbara Vanhoecke holds a PhD in Medical Sciences as well as a Master in Biochemistry. She joined Rousselot in 2017 and is now Innovation Manager Biomedical working in particular on the X-Pure range. Barbara Vanhoecke has published two patent applications and is author/co-author of more than 40 scientific papers.

functions of the body’s extracellular matrix provides the appropriate molecular and mechanical signals needed for the cells to achieve the necessary growth, migration and differentiation.

To generate new, functional and compatible tissue in the transplanted cellular scaffold, low-endotoxin gelatin can provide a favourable environment for cell growth and be used to create hydrogels with the precise characteristics required such as mechanical strength. Since it naturally contains arginine–glycine–aspartic acid (RGD) peptide sequences, gelatin provides attachment sites for cells, which facilitates well-defined mechanical and biological signalling.

2. Why is it important to monitor and regulate endotoxin levels for tissue engineering?

A type of pyrogen (i.e. a substance that induces fever when released into the bloodstream), endotoxins are a component of the exterior cell wall of Gram-negative bacteria. While they do not directly harm any tissue, they can initiate a strong immune response by human immune cells as an indicator for the presence of bacteria. However, they can also trigger other cell types, like stem cells and endothelial cells. Actually, any cell type containing the toll-like receptor-4

is sensitive to these molecules and can therefore be affected in some way. Depending on the concentration and exposure time, endotoxins can negatively impact cellular activity in terms of growth, morphology, differentiation, inflammation and protein expression even at very low levels (<100ng/ml). In stem, immune and endothelial cells specifically, using lowpyrogen gelatin as biomaterial for scaffolds helps to minimise the risks of both immunogenicity and potentially tumorigenicity of the transplanted hydrogel.

When it comes to immunogenicity, it is important to use highly purified biomaterials for the construction of the scaffold. Contamination with elevated levels of endotoxins and other pyrogens creates unwanted immune reactions once implanted in the patient, effectively risking failure of the implant and endangering the patient’s health. Similarly, since (chronic) inflammation can transform cells and induce tumor growth, the use of extra pure biomaterials might also help avoiding deregulated cell growth and thus tumorigenic constructs.

3. What are the benefits of using low endotoxin gelatin in hydrogels?

Gelatin’s biocompatibility and tunable mechanical properties are two essential characteristics in the development of hydrogels.

During the gelation process many parameters, such as temperature, can be changed or controlled in order to achieve the hydrogel structure of interest. Adequate mechanical strength, for example, can be obtained by modifying the crosslinking degree. Also, countless combinations with other molecules (APIs, other biomaterials) are possible with gelatin-based hydrogels.

These properties, combined with its high purity, make low endotoxin gelatin an ideal material for biomedical applications. To qualify the benefits of using low endotoxin gelatins in cellular hydrogels, a study carried out in collaboration between Rousselot and the University Medical Center of Utrecht set out to assess the effect of endotoxins contained in hydrogels on differentiation of mesenchymal stem cells (MSCs) and peripheral blood mononuclear cells (PBMCs). Improved differentiation of MSCs into chondrocytes was seen after 28 days of culturing in a X-Pure low endotoxin gelatin hydrogel, compared to a non-purified gelatin. Also, a reduced inflammatory response of PBMCs was observed when cultured on top of a low endotoxin gelatin hydrogel as evaluated by measuring TNFalpha and CCL-2 release in the culture medium after 3 days of culturing.

X-Pure’s potential in endotoxin-sensitive cell cultures was also highlighted by a second study carried out in collaboration with Ghent University. A comparison on the viability of immune cells (THP-s) in a 10% X-Pure gelatin solution versus MatriGel TM showed a significantly improved cell survival in X-Pure after 3 days of culture. This study highlights the potential of X-Pure as a valid and improved alternative to MatriGel for endotoxin-sensitive cell cultures, as it also represents a solution to the batch to batch variation and the overload of growth factors associated with MatriGel.

Interestingly, gelatin hydrogels devoid from endotoxins can also be used for storage or transport of endotoxin-sensitive cells, as they significantly improve their survival. Preliminary results of a study carried out in collaboration with Ghent University showed that the addition of 10% X-Pure gelatin to the medium significantly improved viability of endothelial cells after 3 days of storage at 4°C, even in absence of serum, and was significantly better compared to non-purified gelatin.

4. What else should hydrogel manufacturers look for when choosing low-endotoxin gelatin?

Among the several factors to be taken into consideration to evaluate low endotoxin gelatins, other pyrogenic activity in the biomaterial used is the first that comes to mind.

These other non-endotoxin pyrogens can also trigger an adverse immune response in the human body and cause an anaphylactic shock. Associating low endotoxin levels to low pyrogenic activity can be a dangerous assumption, as the LAL assay, commonly used to measure endotoxin contamination, does not measure non-endotoxin related pyrogenic activity. The results of Rousselot’s latest purity testing show that the full X-Pure portfolio consisting of both type A and B gelatins (hydrolysed and non-hydrolysed), can be produced and delivered pyrogen-free. This is important since there are no type A gelatins today on the market that are free from pyrogens. For some applications however, such as for the use of gelatin nanoparticles in drug delivery systems, the selection of type of gelatin is very important to get the desired release profile of each compound.

The quality, safety and compliance of excipients can’t be overlooked either. It’s important to partner with a supplier committed to consistently deliver ingredients of the highest quality, compliant with the international standards as well as fully traceable and extensively tested. In such an evolving field, collaborative partnerships with suppliers can be an invaluable source of formulation, technical and R&D support. This is why Rousselot works in close partnership with its customers to co-develop new products and applications.

5. What other exciting applications is X-Pure suitable for?

Highly purified gelatins hold an enormous untapped potential in several emerging application areas. Exciting uses come from the 3D bioprinting sector, for example, where again low endotoxin gelatin can reduce the risk of tissue rejection.

XPure’s low immunogenicity also represents an opportunity to advance innovation in wound dressing applications, as minimising pro-inflammatory stimuli could reduce the risk of sustained inflammation.

Furthermore, the fields of biomolecule and drug delivery are increasingly turning to ultrapure gelatin to prepare injectable micro- and submicron particles as well as colloidal gels that comply with the highest regulation and quality standards