Comparability Studies for Monoclonal Antibody Therapeutics with In Vitro Mode of Action Assays
May 08 2013
Complex protein therapeutics like monoclonal antibodies (mAbs) require comprehensive testing of the biological activity during the whole life cycle to ensure quality, safety and efficacy. For this purpose, functional bioassays reflecting a drug’s mode of action (MoA) are required by the regulatory authorities.
Trends in New Candidate Selection
Most of the current market-approved mAb therapeutics are produced in mammalian cell lines, like CHO, and are mixtures of different glycoforms which are often not fully identical to those of humans. These nonhuman glycoforms may be immunogenic and may cause an enhanced unwanted in vivo clearance. The actual trend is the directed modification of the glycosylation pattern, which is brought to perfection by glycol-engineering with the goal of reduced immunogenicity and higher in vivo effectivity. Thereby the production of partially or completely afucosylated variants of innovator mAb therapeutics is becoming possible. The first candidates of glyco-engineered follow-on mAb therapeutics are already on the market or in late phase clinical trial with promising results.
These new candidate therapeutics require thorough in vitro efficacy testing starting from a very early phase using well-established MoA and binding assays. The described and other modifications often have a significant impact on the in vitro bioactivity determined in MoA assays. These modifications can be used for in vitro biocomparability studies at an early phase during drug development. Even minor differences between the innovator and follow-on biologic can often be detected by direct comparison within in vitro MoA assays.
Rituximab Example
A good example is the comparison of Rituximab innovator to a partially afucosylated Rituximab. The binding capacity of the partially afucosylated mAb to CD20 on the surface of an appropriate target cell line is not affected in a flow cytometry-based binding assay. In contrast to simple binding approaches, MoA assay are capable of reflecting the non-similarity. The ADCC activity is significantly increased and the CDC activity is decreased relative to the Rituximab innovator. The apoptosis activity is slightly enhanced, but this effect is less significant than the effect on ADCC and CDC activity.
Usually a higher in vitro ADCC activity is associated with a potential improvement of the in vivo effectiveness and potency whereas a reduced in vitro CDC activity gives a hint for reduced toxic side effects of the mAb therapeutic.
Conculsion
Taking into consideration that changes in the glycosylation (e.g. partial or complete afucosylation) as well as other modifications on mAb therapeutics do have a significant impact on the in vitro and in vivo efficacy which is reflected by MoA assays, the thorough in vitro testing of newly developed follow-on biologics is becoming more and more valuable. In vitro MoA assays are a powerful tool for the development of biosimilars. A well conducted and documented confirmation of biosimilarity at an early phase allows the reduction of the required clinical work which is usually expensive and time-consuming.
For more information on our in vitro MoA assays, please contact us at askcharlesriver@crl.com.
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