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

Typically, a MCB is sourced from a research cell bank, which may not be GMP-compliant. Thus, before introducing such cells into a GMP facility, minimal biosafety testing (e.g. sterility, mycoplasma, in vitro assay) is required. A MCB is defined as a "homogenous suspension of the cell line (producing the MAb), distributed in equal volumes in a single operation and usually cryopreserved in individual containers for storage."14 It usually consists of 100–200-vial lots.

A WCB is a "homogenous suspension of cells, derived from the MCB at a finite passage level."14 It is prepared using identical conditions and dispensed in identical volumes into cryopreserved vials for storage. Both the MCB and WCB should be stored under controlled (GMP) conditions; the method for preparation, as well as number of vials produced and used, should be recorded and storage conditions documented.

Along with MCB and WCB, post production cells, which are cells propagated up to or beyond the generation number used for routine production, should be tested and characterized.


MAbs derived from the selected cell clone should be precisely analyzed with respect to structural integrity, specificity, and potency using a range of state-of-the-art chemical, physical, immunochemical, and biological tests. To assess biopotency, binding assays and functional in vitro/in vivo assays should be carried out.14,15

When submitting a marketing authorization application, at least six months of stability data should be provided, complemented by further realtime data, which should be supplied as data become available.17


As part of their product analysis, manufacturers must test extensively the cell line used for production. The entire drug development program relies on thoroughly characterized source cells. It is recommended that manufacturers create source cells from nonmanipulated parental cell lines, for instance, via cell fusion or transfection. Based on characterized parental cell line, the presence of adventitious agents—ranging from bacteria, mycoplasma, yeast, and fungi to viruses and transmissible agents—should be addressed in the MCB and WCB.

Contamination of cells may result from infected source animals used for the original cell line.18 Viral contaminants may be introduced by failure of GMP during production or by contaminated excipients. Also, animal-derived raw materials, such as fetal bovine serum or trypsin, may be responsible for the transmission of animal (e.g., bovine or porcine) viruses.19 Regardless of how cells have been propagated, it is necessary to test for bovine and porcine viruses.

Table 2. Summary of tests used for cell bank characterization18, 19
Table 2 summarizes methods for cell bank testing. Each assay is inoculated with a test sample, either a cell lysate or supernatant fluid from cell culture to test for contaminants such as species–specific viruses, adventitious viruses, and retroviruses.

Species–specific viruses are usually detected by administrating the test sample via four different routes into appropriate virus and antibody-free test animals followed by immunological analyses of serum samples for a panel of animal specific viruses. For instance, a murine hybridoma is screened via the aforementioned mouse antibody production(MAP)test for 16 mouse viruses.15 In adventitious agent testing to detect introduced, nonobvious viral contaminants, the test sample is inoculated onto tissue cultures from various species susceptible to a wide range of viruses. Morphological evidence of cytopathology is sought after a suitable incubation period (14 days or 28 days). At the end of the observation period, tests for hemabsorbing and hemagglutination viruses are performed. Retrovirus screening is investigated by reverse transcriptase assay, infectivity assays, and detection and quantification of viral particles by electron microscopy.16,20,21 More test methods are compiled in Table 2. MAbs are considered highly purified products not contaminated by cells; thus, tumorigenicity in athymic mice need not to be addressed.13

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