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

Because residual cellular DNA may be associated with malignant transformation activity, interference with normal gene expression, or production of infectious viruses, the amount of residual DNA in the final bulk product should be quantified. Accepted limits are in the range of 100 pg to 10 ng/dose. A more precise analysis examines not only the residual content, but also the molecular size of the residual DNA; smaller fragments (<500 nucleotides) are not likely to be transforming or oncogenic genes, and are therefore of less concern than larger fragments.30,31

Likewise, the manufacturer needs to assess contaminating host cell proteins (HCPs) and process-related impurities. Risks associated with HCPs relate to adjuvant effects, transforming effects, and allergic and immune reactions. However, limits are not yet established for HCPs.

Other factors, such as route and frequency of administration, may play a role in the occurrence of adverse events.32 During early development, analyzing process impurities and HCPs by generic immunoassays (such as protein A or CHO) may be sufficient. Ultimately, however, in Phase 3 it is necessary to develop customized immunoassays using extracts from both nontransfected and transfected production cell lines.

The final product is subjected to a range of controls as shown in Figure 1. The overall qualification of cell banks and batch-specific safety testing requirements are summarized in the same figure. In Europe, quality specifications for MAbs have recently been integrated in the European Pharmacopoeia. Therefore, manufacturers must demonstrate compliance with Monograph 2031 concerning Monoclonal Antibodies For Human Use, Animal (2031). This does not apply to MAbs produced in vivo, which are governed by the requirements of the relevant authorities.


Fully tested and characterized MCB and WCB can be used as the cell substrate for routine production. In principle, any monoclonal can be produced in vitro by means of cell culture fermentation processes, or in vivo by injecting hybridomas into the abdominal cavity of mice (mouse ascites method). In practice however, certain cases require in vivo production,7 including the following:

  • the production cell line cannot be adapted to in vitro growth conditions
  • the antibody from in vitro culture is prone to denaturation during purification or concentration steps
  • in vitro culture could affect the modification of the molecules (sugar residues) with consequences for the binding and biological activity of the antibody

Table 3. First generation antibodies10,37–38
Some companies have adopted the mouse ascites method for large-scale production of licensed first generation (mouse) antibodies. Table 3 summarizes information about several such murine monoclonals. It should be noted that in most European countries, the ascites method is restricted to certain conditions or even banned36 due to animal welfare considerations.

If the ascites method is justified and authorized, the following regulatory information should be presented in the registration dossier7:

Source Animals

  • information on animals used for production (age, sex, strain, confirmation of specific pathogen free status, source)
  • information on animal health monitoring program (monitoring of stock, mouse-specific viruses, mycoplasma) and demonstration of meeting animal welfare requirements
  • information on physiological and physical examination and testing of animals

Ascites Production

  • information on priming with tumor-promoting compound
  • information on inoculum, cell number, and timing
  • information on ascites harvesting (number, frequency, and procedures)
  • batch definition, storage conditions and duration

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