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Clonality assurance and the rise of therapeutic monoclonal antibody development

Monoclonal antibodies – a recent history

Over three decades ago, in 1986, the first monoclonal antibody (mAb) treatment was approved by the United States Food and Drug Administration (US FDA)[1]. Since then, techniques and technologies to identify, engineer and harness mAbs have advanced and therapeutic antibodies have become the primary focus for many drug developers. The global therapeutic mAb market was valued at approximately US$115.2 billion in 2018 and is expected to generate revenue of $150 billion by the end of 2019 and $300 billion by 20251.

Prior to the development of mAb technologies, antisera (blood serum containing antibodies against specific antigens) from hyperimmunized animals was used to treat infectious diseases such as botulism and diphtheria, and still is in some cases. However, it is now recognized that antibody therapies formulated from species other than humans can cause harmful immune reactions, driving the development ofengineered antibody constructs that carry a lower risk to patients.

Chimeric antibodies were the first engineered improvement where murine constant regions were replaced by human constant regions. Next was the development of the humanization process, resulting in an antibody where only the complementarity determining regions of the variable regions are of mouse-sequence origin. More recently, fully human antibodies have become the new standard, where antigen specificity has been selected either in vivo by the use of genetically modified mice or by antibody engineering processes combined with screening[2].

Therapies based on monoclonal antibodies

While mAbs are widely used in biochemistry, molecular and cellular biology, and medical research, the most beneficial application is their use as therapeutic drugs for the treatment of human diseases, such as cancer, asthma, arthritis, psoriasis, Crohn’s disease, transplant rejection, migraine headaches and infectious diseases. As the use of mAb therapies increases, the list of targets and potential targets is expanding while oncology, immunology, and hematology remain the most prevalent medical applications. Most mAbs have multiple disease indications with at least one that is cancer-related1. Table 1 illustrates the multiple applications and the financial success of the top 10 best-selling monoclonal antibody drugs in 2018.

Top 10 best-selling monoclonal antibody drugs in 2018

Table 1: Top 10 best-selling monoclonal antibody drugs in 20181

Clonality, assurance and the quality of mAb-based drugs

The development, scale-up and eventual manufacture of mAbs requires optimized, stable, productive cell lines to maximize regulatory compliance, safety, patient benefit and economic viability. Crucially, cell lines used for monoclonal antibody production – and any other biologic production – must have demonstrated evidence of clonality. Clonality, or lack thereof, can significantly impact product quality, and as such evidencing clonality is a necessary stage in securing regulatory approval[3].

Over the last decade, Solentim has become the industry ‘go to’ technology for rapid implantation and confident regulatory submission. Solentim’s VIPS™ and Cell Metric® provide high-efficiency single cell seeding and capture the necessary evidence to provide assurance of clonality. Together, they work to accelerate the discovery and development of new biological therapies, enhancing productivity and speed with the highest level of confidence, to help patients receive life-saving therapies faster (see Figure 1).

Chinese Hamster Ovary (CHO) cells

Suitable expression systems for mAbs must be able to deliver high productivity and product quality while maintaining good levels of outgrowth and colony survival. Chinese Hamster Ovary (CHO) cells are the primary choice of mammalian expression system for the production of therapeutic proteins, including mAbs; roughly 70% of all recombinant proteins are made in CHO cells[4].

Since the first use of CHO cells for recombinant protein expression, production processes have steadily improved. Despite other mammalian expression systems being available, CHO cells have clearly been at the center of research on optimizing recombinant protein expression. CHO-based mAb production processes have therefore improved considerably and typically reach the highest product titers with ∼1 g/L in batch and 1–10 g/L in fed-batch processes[5].

Along with cell line optimization, the expression vector, translation efficiency, matrix and growth supplements must also all be optimized to secure the best results.

Technology in action

The race for the next block buster continues at pace. Genmab is Europe’s largest independent biotechnology company, developing antibody-based therapeutics to tackle cancer. Genmab’s cell line development team implemented Solentim’s technology to accelerate its pipeline. “By adopting VIPS and Cell Metric, the CLD team can cut our timelines for transfection to ambr15 from six months down to 10 -13 weeks”, said Jolanda Gerritsen, Technology Expert and Manager of Cell Line Development at Genmab.

The team also increased outgrowth rates and secured proof of clonality; “We can trace back the data to the single cell, select from our top 48 cell lines, and report all the information – our proof of monoclonality is already in there, and that’s really nice”, explained Gerritsen.

Similarly, the Cell Line Development team at Janssen R&D implemented Solentim’s VIPS and Cell Metric CLD for use on its CHO cells lines producing new biologic drugs, which resulted in a 4-6-week reduction in workflow duration. The team also achieved seeding efficiencies as high as 87%, very low incidence of ghost wells (0.06%), and matched the previous outgrowth rate while doubling the number of colonies pre plate compared to manual limiting dilution.

COVID-19

Next generation mAb therapies could take many forms and may provide a possible treatment for COVID-19, with hopeful candidates being expedited for widespread use through mechanisms like the US Food and Drug Administration’s Emergency Use Authorization process. However, mAbs are more difficult to make than many drugs and often are extremely expensive, which means that demand could outstrip supply and many countries might not be able to afford them[6].

Regardless of the exact path that gives rise to the next generation of monoclonal antibody therapies, one thing is for certain: therapeutic antibodies will remain a key feature of the drug development landscape for years to come.

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[1]Lu, R.M., Hwang, Y.C., Liu, I.J., Lee, C.C., Tsai, H.Z., Li, H.J. and Wu, H.C., 2020. Development of therapeutic antibodies for the treatment of diseases. Journal of biomedical science27(1), pp.1-30.
[2]Harding, F.A., Stickler, M.M., Razo, J. and DuBridge, R., 2010, May. The immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the CDR regions. In MAbs (Vol. 2, No. 3, pp. 256-265). Taylor & Francis.
[3]Welch, J.T. and Arden, N.S., 2019. Considering “clonality”: A regulatory perspective on the importance of the clonal derivation of mammalian cell banks in biopharmaceutical development. Biologicals62, pp.16-21.
[4]Li, F., Shen, A. and Amanullah, A., 2010. Cell culture processes in monoclonal antibody production. Pharmaceutical Sciences Encyclopedia: Drug Discovery, Development, and Manufacturing, pp.1-38.
[5]Kunert, R. and Reinhart, D., 2016. Advances in recombinant antibody manufacturing. Applied microbiology and biotechnology100(8), pp.3451-3461.
[6]https://www.sciencemag.org/news/2020/09/provocative-results-boost-hopes-antibody-treatment-covid-19