Cell Lines Development -The Heart of Biologics Manufacturing

As the horizon of biotechnological research expands so does the applications of it in the different biopharmaceutical sector. Rather than starting with a disease and searching its root cause, biologics focuses on detecting genetic variation and finding a therapy that manipulates it. Drugs which are extracted from natural sources, unlike chemically synthesized drugs, are called biologics which are produced by recombinant technology using cell line development methods.

Biologics, such as enzymes, hormones, blood factors, monoclonal antibodies (mAb), allergenics, somatic cells, cell therapies, gene therapy, and tissues, are produced by cutting-edge biomedical research by means of cell line optimization and development procedures. Biologics are not new, development of human growth hormone, insulin, occurred decades ago, but the targets have increased exponentially with the unlocking of new genetic information and a new understanding of subcellular cascades and disease processes. The earlier biologics such as insulin was extracted from slaughtered pigs and cattle, which showed an allergic reaction in many patients. After years of research in engineering a different cell to produce necessary biologics, insulin was produced in 1978 using E. coli with the least allergic reaction. Now the focus is on genetic engineering technology where cell lines produce these products, with more compatibility in human. Efforts are being put forth in optimizing existing host cell systems and alternative host systems, including plants, insect cells, yeasts, and transgenic animals so as to obtain protein/biologic of desired structure, expression, and higher yield. From 2012 to 2018 there are totally 75 FDA approved biologics and they are produced using different cell lines such as CHO, NS0/SP/2/0, E. coli, yeast, HEK293, and others.

At the heart of the biomanufacturing process remains a cell line whose characteristics dictates productivity and product quality of the biologic. The advances in sequencing, genomic and transcriptomic resources for industrially important cell lines coupled with advances in genome editing technology have opened new avenues for cell line development from vector design to cell line screening, have greatly expanded the capability to attain producing cell lines with certain desired traits. According to IQ4I Research, the cell line development service market is estimated to grow at a CAGR of 11.3% to reach $1,519.5 million by 2027. The top companies involved in cell line development are JSR Life Sciences, Lonza, Boehringer Ingelheim, Catalent, Samsung Biologics, and Thermo Fisher Scientific.

The methods of producing a cell line have remained unchanged but the host cells used to derive cell line are often pre-adapted and selected for better traits suitable for industrial manufacturing. The methotrexate (MTX)— dihydrofolate reductase (DHFR) systems and methionine sulfoximine (MSX)—glutamine synthetase (GS) systems have been used to increase the copy numbers of the product genes, consistently yielding very high producing cells. The methods of identifying and isolating these higher producing candidate cells have been refined. A transfected host cell contains an expression vector comprising of a plasmid with an insert of a gene of interest.

There are majorly three cell expression systems: Mammalian expression system, Microbial expression system, and other expression systems. Mammalian cells dominate other cell lines in the production of therapeutic proteins. As they have the capability of performing post-translational modifications, including complex tertiary structure formation through multiple disulfide bonds, glycosylation, and phosphorylation, which are important attributes of many therapeutic proteins. As of December 2018, nearly 57 FDA approved Biologics were produced from mammalian expression systems.

When compared to mammalian cells, bacterial cells such as E. coli provide a higher yield, lower per batch variation, better scalability and lower production cost. But bacteria are not opted most of the time to produce mammalian protein as the protein produced by them may have changes in protein structure due to changes in glycosylation pattern. As of 2018, some of the E.coli produced biologics approved by FDA were Revcovi (Elapegademase-lvlr) and Lumoxiti (Moxetumomab pasudotox-tdfk). Few major companies and their Cell line development technology products are JSR Life Sciences – SUREtechnology platform, Lonza – GS Xceed Gene Expression System, Boehringer Ingelheim – BI-HEX, and Catalent – GPEx.

There have been fewer changes in commonly used host cells which include Chinese hamster ovary (CHO), mouse myeloma NS0 or SP2/0, baby hamster kidney (BHK), and human embryonic kidney (HEK 293) cell lines, showing a mastery over the utilization of the cell lines and reluctance in opting to search for a new one. Although protein glycans produced by CHO and NS0 cell lines differ from each other as well as from those naturally occurring in human proteins, it has not been a major concern, as evidenced by the therapeutic use in humans of proteins produced using both the cell types. CHO is the cell line of choice for biologics development program when compared to other mammalian cell lines because of ease of handling, thorough understanding of its genetics and relatively low cost for development of cell lines. Biologics approved by FDA in 2018 which were developed using CHO and NS0 were Gamifant (emapalumab-lzsg), Libtayo (Cemiplimab-rwlc), Trogarzo (Ibalizumab-uiyk).

Advancement in genomic technology and systematic approach toward designing cells would be a hallmark of the next generation of cell line development. The use of whole genome in Next Generation Sequencing (NGS) to improve clone selection by mapping transgene integration sites and wider application of CRISPR in cell line development and CAR-T (chimeric antigen receptor T-cell therapy) are the future expected strategy in cell line development technology.

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