Maximizing the Potential of Biomanufacturing Cell Lines

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Gary Palmer
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1 Maximizing the Potential of Biomanufacturing Cell Lines

2 Contents Introduction... 3 CHO cells The key trends... 3 The increasing popularity... 3 The biological context... 4 Batch size/price... 4 Novel antibody based therapeutics... 5 Outsourcing approaches... 5 The alternatives to CHO cells... 6 Optimizing CHO cells The key trends... 6 Genome engineering... 6 Methods for improving the GS system... 6 The genome engineering technologies... 7 CRISPR/Cas9 system... 7 recombinant Adeno Associated Virus (raav)... 8 Zinc Finger Nucleases (ZFNs)... 8 Overcoming the challenges in genome engineering... 8 Conclusion... 9 References... 9

3 Introduction The first recombinant protein licensed for use by the United States Food and Drug Administration (US FDA) was human insulin in 1982 (1) ( This was closely followed by tissue plasminogen activator (tpa), the first complex glycosylated protein generated in mammalian cells to be licensed for therapeutic use in Since then, this area of biology has rapidly expanded in the clinic, with an average of 15 new entities being approved by the US FDA every year between 2006 and 2011 for the manufacturing of complex glycoproteins. Although protein yield achieved using CHO cells has increased approximately 100 fold over the years (3), the advances in cell line technology are still limited by the biology of the CHO cell. To increase the potential of the host system, fundamental improvements to the CHO cell line are needed, which is where we believe gene editing can offer a significant step change in biopharmaceutical manufacturing. As the pharmaceutical industry moves towards an ever more fragmented market due to the establishment of personalized medicine, smaller manufacturing runs need to become economically viable. Equally, as therapeutics based around novel protein architecture are developed that are not efficiently produced in current systems, modification of these systems are required to maximise the benefits derived from these exciting new therapies. CHO cells The key trends The increasing popularity The natural characteristics and adaptability of CHO cells have contributed to their popularity Although many systems have been developed to generate biologics over the past 25 years, prokaryotic systems are still primarily used for simple proteins that do not require complex glycosaccharides for their efficacy or stability, and for biologics that need these complex post translational modifications, a mammalian host is needed. CHO cells have recently become the predominant expression system. More recently the safety implications of using animal derived products in the culturing of production cells has led to a preference for cells that are grown in serum free conditions in chemically defined media. Concomitantly, the manufacturing advantage conferred by using cells that can grow at high density in suspension culture has led to a preference in the industry for these cells. The natural characteristics of CHO cells have contributed to their popularity. Amongst others, they: Are relatively easily adaptable to suspension growth in animal component free, chemically defined media, Express and secrete recombinant protein (including antibodies) at relatively high levels and; Provide a human like glycosylation profile. The glycosylation profile is particularly important, preventing the activation of an immune response which can lead to unwanted side effects or the rapid clearance of the drug. In comparison, murine cells for example can induce immunogenic effects through the incorporation of an alpha gal

4 residue (4). Once stably transfected with a vector expressing recombinant protein they generate a heterogeneous population of cells, some of which are expressing high levels of the protein of interest through a combination of integration sites and the ability of CHO cells to amplify regions of genomic DNA. The biological context Advances in cell line technologies are significant, but are still limited by the natural biology of the CHO cell Extensive optimization of culture media and feeding strategies are such that expression levels have increased approximately 100 fold over the initial capabilities. In combination, there have been some alterations to the CHO cells themselves to decrease the time taken to identify the highest expressing clones through the development of metabolic selection systems. These modifications have focussed on selecting those cells which have integrated the expression cassette into sites leading to high expression of recombinant product. Initially this was achieved through the use of the Methotrexate/Dihydrofolate Reductase (MTX/DHFR) system, and then more recently the use of the Methionine Sulphoximine/Glutamine Synthetase (MSX/GS) system. The GS selection system is more stringent than DHFR, leading to reduced timelines to identify high expressing clones (5). However, these advances in cell line technology are still limited by the natural biology of the CHO cell. A selection system can only identify those clones naturally expressing higher levels of protein, and media optimization can only ensure that those clones selected are in the best possible health to produce maximal amounts of protein. To increase the potential of the host system, something more fundamental needs to be improved. Batch size/price It is a valid question to ask whether further advancement is necessary. After all, titres are higher than ever and the time to isolate the production clone is now measured in weeks instead of months or years. Despite this, treatment courses of biotherapeutics are more than twenty times more expensive than small molecule therapeutics (6) ( rch/opportunities for biosimilardevelopment). The CHO expression system is being pushed to its limits as manufacturers drive to make smaller batches economically viable to keep pace with the development of ever more personalized approaches to medicine. Figure 1 Patients are stratified into ever smaller groups as mechanisms of disease progression are linked to individual genotypes, leading to improved therapeutic outcomes for different groups of patients. The same economic argument above can be applied to orphan diseases, with clear benefits to sufferers of these relatively rare conditions.

Novel antibody based therapeutics There is an increasing shift towards novel classes of antibody based therapeutics despite setbacks The production of monoclonal antibodies in CHO cells has been the

5 Novel antibody based therapeutics There is an increasing shift towards novel classes of antibody based therapeutics despite setbacks The production of monoclonal antibodies in CHO cells has been the focus of many recent therapies, and the expression of these therapeutics at multi g/l levels is becoming the industry norm. However, even with the increased potency afforded by the new technologies, expression levels are often not high enough to make the production of the active protein economically viable. Figure 2 There is an increasing trend towards novel classes of antibody based therapeutics Existing therapies could also benefit from improving the manufacturing platform. The regulatory acceptance of biosimilars and the large potential markets produced as patents of successful drugs expire have led to a dramatic expansion in companies entering this field. As the current estimated cost of a biosimilar is approximately 50% of the cost of the originator product, potential savings in manufacturing costs are attractive to companies in this field. When producing a biosimilar, similarity of post translational modifications to the originator product is one of the most challenging aspects to address. Currently this is achieved through process modification, but control over aspects such as glycosylation would be a beneficial feature of a production platform. Outsourcing approaches Outsourcing is becoming increasingly popular in the industry with trends towards decreased production scales and increased flexibility While some companies have stated they will keep manufacturing of complex products in house (7) ( Processing/Novartisto keep complex biologics manufacturing inhouse), there is a trend in the industry towards outsourcing manufacturing (8) ( d Articles/ Biopharmaceutical Outsourcing Continues to Expand/). This has led to the emergence of many Contract Manufacturing Organisations (CMOs) with expertise in biologics manufacturing who can fill this need. These companies require efficient use of flexible facilities in order to maximize the utilisation of existing capacity. Multiple smaller bioreactors are now the norm in new facilities to meet market need instead of the large legacy plants containing tens of thousands of litres of capacity. This has also been enabled by the increased adoption of Single Use Bioreactors (SUBs) instead of the investment required to establish large stainless steel bioreactors. The same logic holds for small biotech companies wishing to keep manufacturing in house. The natural extension of this is a trend towards decreased production scales in the industry (9). If cell lines are capable of generating high yielding clones in order for the required amount of protein to be produced at decreased scale, this would fit with the movement towards smaller scale production. Similarly, decreasing the run times of

6 bioreactor batches would increase the flexibility and capacity of existing plants. It is therefore clear that improved production systems leading to increased manufacturing efficiency or control over post translational modifications is attractive to a variety of manufacturers with differing priorities. The alternatives to CHO cells There are a number of concerns that limit uptake of other novel systems To achieve this, the existing options are to alter the CHO cell platform to allow improved expression, or to change systems altogether for one that has an improved basal capacity. There are a number of different available systems, each with their own characteristics. For example plant cells scale easily and produce high titres, while insect cells have also shown high titres. However, there are a number of concerns that limit uptake of novel systems. From a practical perspective, these systems are competing with an extensive history of production of safe therapeutics, so there are concerns over nonmammalian glycosylation moieties. There is similarly a large body of experience of manufacturing using CHO cells. This means that the system is incredibly well characterized, with a known safety profile and well established processes to ensure product quality and consistency between batches. There is therefore a strong case for the industry not to move away from CHO cells but instead fundamentally improve this existing system. There are also many more scientists trained in handling CHO cells than any other expression system, so a change in system would require significant time and resource investment. Optimizing CHO cells The key trends Genome engineering CHO cells are becoming the system of choice with genome engineering allowing almost any characteristic to be altered At Horizon, we are of the opinion that there is a great opportunity to revolutionise the current CHO platform by altering the genome, and we have begun an extensive program to achieve this. This avenue of research could have significant implications for the future of bioprocessing. The GS system is the current industry standard selection system Given the correct targets, the ability to engineer the genome to improve the CHO cell expression system has almost limitless potential. The GS system is the current industry standard selection system and in order to encourage industry uptake and innovation, access to these cells is becoming easier through changes to the licensing paradigm. These cells will then be targeted through further engineering to remove the need for a completely alternative system. Methods for improving the GS system Genome engineering is enabling specific panels of cell lines to overcome particular challenges In order to improve on the GS system, a number of different approaches can be taken, with a number of different steps that could benefit from intervention. Potential modifications can include:

Whole gene knockouts, Whole gene knock ins and; Individual point mutations on target genes. with all of the above affecting the activity of target enzymes or substrates.

7 Whole gene knockouts, Whole gene knock ins and; Individual point mutations on target genes. with all of the above affecting the activity of target enzymes or substrates. Similarly, multiplexing many different engineering events within one line in a logical way will have synergistic effects on the target phenotype. recombinant Adeno Associated Virus (raav) and; Zinc Finger Nucleases (ZFNs) CRISPR/Cas9 system Genome engineering is now more accessible than ever before with CRISPR/Cas9 In the future, it will even be possible to introduce entire new molecular pathways For example, one could reasonably anticipate that manipulating both transcription and translation would have a multiplicative effect on specific productivity. Alternatively regulation of product quality could be finetuned by controlling transit through the ER as well as careful modification of chaperone proteins. In the future, it will even be possible to introduce entire new molecular pathways. Trait stacking these improvements onto an existing cutting edge line will encourage broad uptake through the industry. By encouraging industry wide uptake of these cells through licensing and performance, they will become a platform technology leading to further improvements through innovation and optimization within different aspects of process development. Furthermore, innovation through engineering these high performance cells for specific applications will lead to a panel of different cell lines, each optimized to overcome particular challenges. The genome engineering technologies Genome editing technologies are available in order to begin to mine the potential of cell line engineering, including: Figure 3 The excitement that has followed the discovery of the CRISPR/Cas9 system (13) has led to a surge in interest in engineering technologies This system has much potential in improving the stacking of engineered modifications, and has already been used to multiplex engineering events (14). Indeed, due to its relatively recent discovery it is still rapidly evolving, with improvements being made to the specificity and efficiency of this exciting new technology, which is making genome engineering more accessible than ever before. This has also been enabled through the sequencing of the CHO genome allowing efficient targeting vectors to be designed. CRISPR is used to rapidly screen hundreds of potential targets in order to identify those with significant effects, and to rapidly test multiplexed edits for synergistic effects It must be stressed that at this stage, the Intellectual Property surrounding CRISPR engineering has yet to be resolved, so while the technology has much potential at the current time, its use is restricted to Research and Development. CRISPR/Cas9 system,

Figure 5 ZFNs, care must be taken to avoid unwanted mutations leading to unintended consequences. Figure 4 raav technology Induction of homologous recombination can be achieved.

8 recombinant Adeno Associated Virus (raav) Although an established technology it can be time consuming specificity concerns (16). Aside from this, access to ZFNs are cost prohibitive to the majority of companies for use in biomanufacturing. Figure 5 ZFNs, care must be taken to avoid unwanted mutations leading to unintended consequences. Figure 4 raav technology Induction of homologous recombination can be achieved. As the nature of raav means that only one allele can be targeted at a time, the process of introducing multiple events can be time consuming. To overcome this, CRISPR is used to rapidly screen hundreds of potential targets in order to identify those with significant effects, and to rapidly test multiplexed edits for synergistic effects. Following target validation, raav can then be used to generate cell lines that can be used in commercial manufacturing. Zinc Finger Nucleases (ZFNs) Lower efficiencies and specificity concerns currently limits its popularity Zinc Finger Nucleases (ZFNs) work on similar principles to the CRISPR/Cas9 system but have lower efficiencies than CRISPR (15) ( Articles/ Genome Editing with CRISPRs TALENs and ZFNs/) as well as Another area that could also have a large impact on the manufacture of biotherapeutics is in antibody engineering. It is becoming apparent that substitutions of only a few amino acids in the antibody sequence can have a large impact not only on potency, but also on expression and aggregation. Similarly, modification of signal peptides can have a marked increase in the levels of secreted protein. While a full discussion on the potential of this area is beyond the scope of this article, combining optimization of the cell line with co developed expression vectors could have a significant impact on the field in the future. Overcoming the challenges in genome engineering Increased understanding of CHO biology through interpretation of the available omics information is enabling the industry to identify the best targets While the potential of cell line engineering is almost endless, the practicalities are currently limiting. Not only do the best phenotypes need to be identified, but the correct targets then need to be established. This has recently been enabled by the increase in the availability of omics data improving our

understanding of CHO biology (10 12). There is still much to be done in order to generate a better understanding of the implications of this new information.

9 understanding of CHO biology (10 12). There is still much to be done in order to generate a better understanding of the implications of this new information. Mathematical modelling is rapidly evolving, which will have a positive effect on the ability to predict the impact of a particular modification, but investment in bioinformatics is needed to keep pace with the ambitions of the industry. Due to the ability of a CHO cell to adapt to different pressures, many different proteins in one cell will need to be modified before a strong effect is seen. For example, the complex nature of the metabolic network means that there is a lot of redundancy in the system, and modification of many different aspects may need to occur to achieve the desired result. Conclusion In conclusion, at Horizon, we believe that cell line engineering has great potential to improve existing CHO cell expression systems in a number of ways beyond what is currently available. This is not a simple enterprise, and not without its risks, but the reward could be the ability to produce biotherapeutics cheaply enough to make not only existing therapies more affordable, but allow small production runs to allow the possibility of personalized medicine to become a reality. References 1. A History of Firsts. 2. Lai T, Yang Y, Ng S. Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production. Pharmaceuticals. Multidisciplinary Digital Publishing Institute; 2013;6(5): Hacker DL, Nallet S, Wurm FM. Recombinant protein production yields from mammalian cells: past, present, and future Figure 6 Horizon s GS / cell platform is the foundation from which it is launching the revolution for the bioprocessing industry. We are now at the stage when increased understanding of CHO biology through interpretation of the available omics information allows us to identify the best targets. Equally, once the correct targets and modifications have been identified, the ability to introduce these to the cells is limitless. 4. van Beers MMC, Bardor M. Minimizing immunogenicity of biopharmaceuticals by controlling critical quality attributes of proteins. Biotechnology Journal. WILEY VCH Verlag; 2012;7(12): Fan L, Kadura I, Krebs LE, Hatfield CC, Shaw MM, Frye CC. Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells. Biotechnology and Bioengineering. Wiley Subscription Services, Inc., A Wiley Company; 2011;109(4): Opportunities for biosimilar development. 7. Stanton D. Novartis to keep complex biologics manufacturing in house

10 8. Langer E. Biopharmaceutical Outsourcing Continues to Expand. Pharmaceutical Outsourcing. 2014Jan Jagschies G. Where is Biopharmaceutical Manufacturing Heading? BioPharm International Meuris L, Santens F, Elson G, Festjens N. GlycoDelete engineering of mammalian cells simplifies N glycosylation of recombinant proteins. Nature Biotechnology. Nature Publishing Group; 2014;32(5): Brinkrolf K, Rupp O, Laux H, Kollin F, Ernst W, Linke B, et al Chinese hamster genome sequenced from sorted chromosomes. Nature Biotechnology. Nature Publishing Group; 2013;31(8): Lewis NE, Liu X, Li Y, Nagarajan H, Yerganian G, O'Brien E, et al Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome. Nature Biotechnology. Nature Publishing Group; 2013;31(8): Kildegaard HF, Baycin Hizal D, Lewis NE, Betenbaugh MJ. The emerging CHO systems biology era: harnessing the omics revolution for biotechnology. Current Opinion in Biotechnology. 2013;24(6): Mali P, Esvelt KM, Church GM. Cas9 as a versatile tool for engineering biology. Nature Methods. Nature Publishing Group; 2013;10(10): Grav LM, Lee JS, Gerling S, Kallehauge TB, Hansen AH, Kol S, et al One step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment. Biotechnology Journal. WILEY VCH Verlag; 2015;:n/a. 15. Perkel J. Genome Editing with CRISPRs, TALENs and ZFNs Pattanayak V, Ramirez CL, Joung JK, Liu DR. Revealing off target cleavage specificities of zinc finger nucleases by in vitro selection. Nature Methods

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Course Agenda. Day One Course Agenda BioImmersion: Biotech for the Non-Scientist A three-day, in-depth course that provides the background required for understanding today s fast-paced biotech marketplace. Beginning with an