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 hybridoma cell lines used as fusion partners, manufacturers must detail the following:

  • source species, strain, and tissue
  • feeder cell line
  • immortalization procedures
  • nature of the immunization antigen
  • immunization procedure
  • screening and selection processes
  • cell cloning
  • if applicable, adaptation to serum-free medium.

If a monoclonal is expressed in genetically engineered cells such as CHO, the origin, isolation, and cloning of antigen-recognizing DNA regions into the expression vector should be described.22, 23 The registration documentation should also contain the construction of the cell line, including vector and transgene nucleotide sequences, transfection procedures, cloning, amplification, and as the case maybe the change to serum-free medium should be described.

In addition, the genome structure should be investigated and stability of the cell line, copy number, and location of the transgene on chromosomes demonstrated. Species should be verified on the chromosome number or banding level by means of karyology.

It is important to note that after the cell substrate has been developed, it is not easy to switch to another cell line for production. If the cell line must be changed, for instance, to increase yield—comparability between the old and new cell banks has to be shown. This may not be limited to analytical data: depending on the development stage, nonclinical and clinical bridging studies may be required to show bioequivalence, safety, and efficacy.24

Culture and Harvest

Cells can be cultured up to a defined number of passages, followed by recovery of product in a single harvest. Another option is multiple harvests during continuous growth of cells and cultivation for defined period, which requires careful surveillance of the cell line throughout its life span.

Figure 1
Each harvest of unprocessed bulk should be monitored by in-process testing for product potency, bioburden, endotoxin, and mycoplasma. Furthermore, for single harvest processes, representative samples of at least three lots of fermenter supernatant should be subjected to comprehensive biosafety testing14 as shown in Figure 1.


A downstream purification process is designed to enrich the product and to eliminate contaminating proteins, DNA, endotoxin, process-related components (media constituents, leachables), viruses, and prions. Process development hinges on finding the most efficient route from the starting material to pure, active final product, considering both yield and viral clearance. A characteristic purification process consists of a primary recovery step followed by multiple adsorption and chromatographic operations and final polishing steps (e.g., size-exclusion chromatography). In many cases, antibody is captured onto a Protein A matrix, followed by low-pH hold and elution, which is effective as a combined purification and virus inactivation step.27

Viral Clearance

The purification process should include at least two robust viral clearance steps that inactivate or remove viruses based on different mechanisms (e.g., inactivation by low-pH treatment and nanofiltration). The capacity of a manufacturing process to reduce, inactivate, or eliminate viruses and other contaminants must be demonstrated by virus validation studies. In designing such studies, researchers take into account the nature of the cell line-in particular, the presence of cell-line-specific endogenous retroviruses and other relevant viruses.28, 29

Final Bulk or Finished Product

Purified bulk product is formulated and aseptically filled into final container (e.g., prefilled syringe, infusion bottle). Normally, purified bulk itself is tested for sterility and residual DNA before formulation and preparation of finished product.

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