Upstream Processing: Regulatory Considerations Regarding Quality Aspects of Monoclonal Antibodies - An alternative to the hybridoma method is producing engineered antibody molecules and fragments


Upstream Processing: Regulatory Considerations Regarding Quality Aspects of Monoclonal Antibodies
An alternative to the hybridoma method is producing engineered antibody molecules and fragments by in vitro methods.

BioPharm International
Volume 20, Issue 7

For large-scale in vivo production a high number of mice (up to several thousand) may have to be used.38 Following priming and inoculating with selected hybridoma cells (e.g., WCB), mice need to be monitored daily as a process control measure. About 5 mL ascites per mouse is collected approximately two to four weeks post inoculation.39 Once a batch is harvested, ascites are tested for a range of parameters (potency, bioburden, endotoxin, mycoplasma, murine viruses) and antibody is purified as described previously.


Table 4. Examples of cell lines used for production of MAbs34–35, 41–43
MAbs should preferably be manufactured by in vitro methods to avoid using animals. As indicated, both mouse cell lines and genetically engineered mammalian cell lines have been used in producing licensed antibodies. For stable expression of recombinant MAbs in continuous mammalian cell lines, such as CHO K1 or SP2/0 (a murine myeloma cell line), are generally preferred. Productivity of selected continuous cell lines (CCLs), which are either cultivated as attached cells on carrier materials or in suspension in bioreactors, is often further increased and maximized by vector amplification.12,18,40 CCLs enable manufacturers to establish cell banks for a reliable production source based on standardized and characterized cells. Also, they are easier to cultivate than primary or diploid cells, require only simple culture media, and can be adapted to serum-free medium. Some cell substrate examples from licensed MAbs are given in Table 4.

For large-scale production, antibody product is usually enriched from cell-free supernatant from up to 15,000 L fermenters (e.g. Synagis: 10,000 L, Herceptin: 12,000 L,35,43) and purified as described above.


CCLs have been isolated by serial subcultivation of primary animal cells, transformation of normal cells with oncogenic virus or, as in the case of hybridomas, by fusion of tumor and B-cells.31 Concerns about the safety of products derived from such cell lines relate particularly to the presence of the following:

  • human pathogenic viral contaminants and potential transmission of viruses to humans
  • tumorigenic or mutagenic residual cellular DNA
  • antigenic or unwanted catalytic (e.g., growth-promoting) HCPs

Despite these concerns, the safety of products derived from established CCLs (e.g., CHO) is broadly accepted, and well-characterized standard cell lines are available.31 In terms of a particular production cell line, biosafety is achieved by the following complementary approaches:18,19

  • Rigorous control of cell lines by testing or characterization and control of raw materials used during manufacture
  • in-process and finished product testing
  • virus clearance and reduction steps in the manufacturing process
  • strict adherence to GMP

In order to minimize viral risks during propagation of CCLs, it is important to source serum from reliable suppliers who provide certificates of analysis demonstrating freedom from viruses in compliance with the relevant guidance (e.g., CPMP/BWP/1793/02). Furthermore, compliance with TSE guidance (e.g., 2004/C 24/03) must be shown. In addition to established cell lines (e.g. CHO, NSO), other cell lines of human (e.g., PerC6) or insect (e.g., Sf9) origin may be used pending a comprehensive biosafety assessment.

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