A central pillar of the biotechnology and pharmaceutical industries continues to be the development  of biological drug products manufactured from engineered non-mammalian cells and mammalian cell  lines or human derived cell lines. Cell line development has emerged as a cornerstone in the  biopharmaceutical industry, significantly influencing the speed, quality, and efficiency of therapeutic  protein production. Regulatory flexibility regarding cell line selection for biosimilars has spurred  innovation in this area. Modern advancements, including high-throughput screening, single-cell  cloning, and genome editing, have accelerated development timelines while ensuring product purity  and potency. The industry is expanding beyond traditional CHO cell lines to explore diverse host  systems, each offering unique advantages. Comprehensive cell line characterization, enabled by  advanced analytical tools, is crucial for maintaining product quality and regulatory compliance. As the  industry transitions towards continuous processing, cell line robustness becomes paramount.  Ultimately, these advancements are driving the development of superior biotherapeutics, addressing  unmet medical needs, and shaping the future of the industry.^[1]

In the guidance document ‘Scientific Considerations in demonstrating biosimilarity to a reference  product’ dated 28 April 2015, FDA states that “By contrast, the manufacturer of a proposed product is  likely to have a different manufacturing process (e.g., different cell line, raw materials, equipment,  processes, process controls, and acceptance criteria) from that of the reference product and no direct  knowledge of the manufacturing process for the reference product.” This goes on to say that the  sponsor of a biosimilar program might not have knowledge about the cell line used by the  manufacturer of the reference product and hence need not use the same cell line, as long as safety,  purity and efficacy of the proposed biosimilar is comparable with the reference product. In a nutshell,  the sponsors of biosimilar programs can consider using a host cell line different from what is used by  the manufacturer of the reference product provided the host cell has traceable clean history,  demonstrated safety in humans and provides advantages over what is used for manufacturing the  reference product. Innovative advancements in cell line development have revolutionized the  biopharmaceutical landscape. This crucial process involves creating cell lines that produce therapeutic  proteins and has witnessed an explosion of new technologies and methodologies. These  developments promise to accelerate drug discovery and ensure that the produced biologics are of  superior quality and efficacy.^[2]

 In earlier times, bacterial expression systems, yeast expression systems (e.g. Saccharomyces cerevisiae and Pichia pastoris) achieve rapid cell growth and high-protein yields with straightforward production  scalability and without the need for animal-derived growth factors. The key challenge associated with  yeast expression systems is their production of high mannose residues within their expressed PTMs  (50–200 vs three molecules in human cells, as part of either N- or O-linked glycan structures), which  may confer a short half-life and render proteins less efficacious and even immunogenic in humans. The  majority of currently licensed biotherapeutic products are produced in non-human mammalian  expression systems, as these systems are able to produce PTMs that (outside of a human expression system) most closely resemble those in humans. These expression systems are used to produce mAbs,  hormones, cytokines, enzymes and clotting factors.^[3]

Human tissue plasminogen activator (TPA) was the first marketed biotherapeutic expressed in CHO  cells and the CHO-DUXB11 (double knockout gene dihydrofolate reductase (DHFR) gene) line was used  to produce hundreds of kilograms of protein. Human tissue plasminogen activator (tPA) is classified  as a serine protease (enzymes that cleave peptide bonds in proteins). It is thus one of the essential  components of the dissolution of blood clots. Examples of these drugs include alteplase, reteplase,  and tenecteplase given under monitored administration to patients with myocardial Infarction  percutaneous transluminal coronary angioplasty, pulmonary embolism and thrombolysis (e.g., deep  vein thrombosis). Alteplase is the normal human plasminogen activator and is FDA-approved for  managing patients with ischemic stroke, myocardial infarction with ST-elevation (STEMI), acute  massive pulmonary embolism, and those with central venous access devices (CVAD). Reteplase is a  modified form of human tPA with similar effects but a faster onset and longer duration of action. It is  currently FDA-approved for the management of acute myocardial infarction. Preferred over alteplase  due to its longer half-life, allowing it to be given as a bolus injection rather than through an infusion  like alteplase. Tenecteplase is another modified version of tPA with a longer half-life. Its indication is  the management of acute myocardial infarction. These drugs are administered intravenously.  Erythropoietin (EPO) is a growth factor produced in the kidneys that stimulates the production of red  blood cells. It works by promoting the division and differentiation of committed erythroid progenitors  in the bone marrow. Epoetin alfa (Epoge) was developed by Amgen Inc. in 1983 as the first rhEPO  commercialized in the United States, followed by other alfa and beta formulations. Epoetin alfa is a  165-amino acid erythropoiesis-stimulating glycoprotein produced in cell culture using recombinant  DNA technology and is used for the treatment of patients with anemia associated with various clinical  conditions, such as chronic renal failure, antiviral drug therapy, chemotherapy, or a high risk for  perioperative blood loss from surgical procedures. It has a molecular weight of approximately 30,400  daltons and is produced by mammalian cells into which the human erythropoietin gene has been  introduced. The product contains the identical amino acid sequence of isolated natural erythropoietin  and has the same biological activity as the endogenous erythropoietin. Epoetin alfa biosimilar, such as  Retacrit (epoetin alfa-epbx or epoetin zeta), has been formulated to allow more access to treatment  options for patients in the market. The biosimilar is approved by the FDA and EMA as a safe, effective  and affordable biological product and displays equivalent clinical efficacy, potency, and purity to the  reference product. Epoetin alfa formulations can be administered intravenously or subcutaneously.^[4]

CHO cell linesare the industry standard for biotherapeutic protein production due to their ability to  produce complex human-like protein modifications. Their long-standing use has led to extensive  knowledge and established processes, accelerating development and manufacturing timelines.  However, CHO cells have limitations in producing specific human glycosylation patterns and introduce  foreign glycans (α-gal and NGNA), potentially increasing immunogenicity. Other cell lines, such as BHK,  and historically, murine myelomas (NS0, Sp2/0), have been employed, but CHO cells generally offer  superior protein quality and process efficiency. While CHO cells remain the dominant choice, ongoing  research and development aim to address their limitations and explore alternative expression systems  to optimize biotherapeutic product development.^[5]

Human cell lines are superior to other mammalian systems for producing proteins that closely mimic  those found in the human body. This advantage is particularly evident in their ability to accurately  replicate the complex sugar molecules (N-glycans) attached to proteins, which significantly impact a  protein's function, stability, lifespan, and immune response. To enhance protein quality and reduce  immune reactions, researchers have developed new human cell lines like PER C6 and CAP. Several  biotherapeutic products made using these and other human cell lines, including HEK293 and HT-1080, are currently under investigation. HEK293 cells, in particular, have a long history of producing proteins  for research and have been approved for manufacturing five therapeutic drugs. These agents are  drotrecogin alfa (XIGRIS?; Eli Lilly Corporation, Indianapolis, IN), recombinant factor IX Fc fusion  protein (rFIXFc; Biogen, Cambridge, MA), recombinant factor VIII Fc fusion protein (rFVIIIFc; Biogen,  Cambridge, MA), human cell line recombinant factor VIII (human-cl rhFVIII; NUWIQ?; Octapharma,  Lachen, Switzerland) and dulaglutide (TRULICITY?; Eli Lily, Indianapolis, IN). Drotrecogin alfa is a  recombinant activated protein C that was approved by the FDA in 2001 and by the EMA in 2002 for the  treatment of patients with severe sepsis. HEK293 cells were chosen by the manufacturer for  production of drotrecogin alfa because its activity required two PTMs, propeptide cleavage and γ- carboxylation of its glutamic acid residues, which CHO cells cannot produce with adequate efficiency.  The product was approved but was later voluntarily withdrawn from the market by its manufacturer  (Eli Lilly) in 2011 following the randomized placebo-controlled Prospective Recombinant Human  Activated Protein C Worldwide Evaluation in Severe Sepsis and Septic Shock (PROWESS-SHOCK) trial,  which demonstrated no mortality benefit with drotrecogin alfa compared with placebo for patients  experiencing septic shock. Therefore it was later withdrawn due to lack of efficacy in clinical trials  despite being successfully produced in HEK293 cells to overcome limitations of other cell types.^[6] 

Cell line development stands as a pivotal force propelling advancements in the biopharmaceutical  industry. By optimizing protein production, enhancing product quality, and accelerating development  timelines, it has become an indispensable component of modern drug discovery and manufacturing.  While CHO cells have historically dominated the field, the pursuit of superior protein characteristics  and the expanding biosimilar market are driving the exploration of alternative cell lines and innovative  cultivation methods. The convergence of technological breakthroughs, such as genome editing and  high-throughput screening, with a deep understanding of cell biology is poised to unlock new frontiers  in biotherapeutic development, ultimately improving patient outcomes. As the industry continues to  evolve, the strategic development and optimization of cell lines will remain a cornerstone for the  creation of safe, efficacious, and accessible medicines.

Reference: 

1. Zhu MM, Mollet M, Hubert RS, Kyung YS, Zhang GG. Industrial Production of Therapeutic Proteins:  Cell Lines, Cell Culture, and Purification. Handbook of Industrial Chemistry and Biotechnology. 2017  May 3:1639–69. doi: 10.1007/978-3-319-52287-6_29. PMCID: PMC7121293.

2. Sreenath Kadreppa; Host Cell Line Selection for Biosimilar Product Development; LinkedIn Post date: May 3, 2018 accessed date Aug 01,2024. URL: https://www.linkedin.com/pulse/host-cell-line  selection-biosimilar-product-sreenath-kadreppa-ph-d/.

3. Dumont J, Euwart D, Mei B, Estes S, Kshirsagar R. Human cell lines for biopharmaceutical  manufacturing: history, status, and future perspectives. Crit Rev Biotechnol. 2016 Dec;36(6):1110-1122.  doi: 10.3109/07388551.2015.1084266. Epub 2015 Sep 18. PMID: 26383226; PMCID: PMC5152558. 

4. Majidzadeh-A K, Mahboudi F, Hemayatkar M, Davami F, Barkhordary F, Adeli A, Soleimani M,  Davoudi N, Khalaj V. Human Tissue Plasminogen Activator Expression in Escherichia coli using  Cytoplasmic and Periplasmic Cumulative Power. Avicenna J Med Biotechnol. 2010 Jul;2(3):131-6.  PMID: 23408156; PMCID: PMC3558155. 

5.Alison Halliday, PhD ; Advances in CHO Cell Line Development for Biotherapeutics; Biopharma  Published on : November 17, 2023; Accessed on: Aug 01,2024. URL: https://www.technologynetworks.com/biopharma/articles/advances-in-cho-cell-line-developmentfor-biotherapeutics-381065.

6. Gupta S et.al. Engineering protein glycosylation in CHO cells to be highly similar to murine host  cells.Front. Bioeng. Biotechnol., 16 February 2023 Sec. Bioprocess Engineering Volume 11 - 2023 doi: https://doi.org/10.3389/fbioe.2023.111399.

About the Author:

Shruti  Talashi    

Ms.  Shruti  Talashi  boasts a dual mastery of lab research and writing.  Her doctoral study outcome as M.Phil in biomedical science while studying  breast cancer and an extraordinary masters degrees dissertation work on  exploring role of Gal-lectin in cancer metastasis fuels her extensive  research interests. She has gained few publication in journals. Bridging the  science-public gap is her passion, aided by expertise in diverse techniques.  From oncology to antibiotic/drugs production, she's led and managed complex  projects, even clinical trials. Now, as a freelance Content Coordinator for  Sinoexpo Pharmasource.com, her industry knowledge shines through valuable  insights on cutting-edge topics like GMP, QbD, and biofoundry.