Glycosylation and microheterogeneity

Humanized mAbs are produced in bioreactors that use cell lines cloned to express the desired product. While the fidelity of the amino acid backbone of the humanized mAbs is excellent, the control of posttranslational modifications, such as glycosylation, is not. Minor modifications of the manufacturing process, many unintentional, can produce dramatic differences in the microheterogeneity or distribution of glycoforms in the expressed protein. Different glycosylation patterns can dramatically affect mAb PK and PD because glycosylation appears to protect the protein from digestion by endogenous proteases, thus resulting in a different half-life and potentially different efficacy.

Pharmacokinetics: safety and effficacy

Figures 4–5: Possible free small molecules can be derived in vivo from the following precursors: (1) monoclonal antibody (mAb)–linker–drug goes to mAb–linker + drug; (2) mAb–linker–drug goes to mAb + linker–drug then goes to linker + drug; (3) mAb–linker–drug gets peptide degraded to peptide–linker–drug, which can go to peptide–linker + drug or to peptide + linker–drug, which then goes to linker + drug. ADC is antibody–drug conjugate. (FIGURES 4 AND 5 ARE COURTESY OF THE AUTHORS)

The most complete assessment of plasma exposure during the development of an ADC includes the bioanalytical measurement of four critical attributes:

• The amount of free drug released from the ADC into circulation; this assay is relatively straightforward but requires method development and validation to ensure that no additional free drug is produced during execution of the assay.
• The amount of drug that is still attached to the humanized mAb, which is usually determined by either filtration or immune-precipitation of the ADC from a plasma sample, followed by chemical release of the small molecule from the protein, with subsequent LC–MS/MS analysis of the small molecule to yield the "bound-drug" amount.
• The amount of free antibody, which is determined using an immunoassay that is specific for the antibody but does not cross-react with the ADC. Often an assay is developed where all human IgGs are isolated by immune-precipitation specific for an intact Fc portion, followed by an ELISA specific for the idiotype.
• The ADC complex, which can be measured using an ELISA assay specific for the ADC that does not cross react with free antibody. The reagents for this assay are very difficult to develop because the assay requires an antibody that will recognize the linker-small-molecule complex as the epitope. Alternatively, these assays may involve digestion followed by LC–MS/MS detection, or, if the small molecule is attached to the Fc portion of the ADC, an immunoprecipitation of the Fc portion could separate the ADCs from the free antibody, followed by detection with an anti-idiotype ELISA assay.

Although this set of assays seems comprehensive, the lack of information regarding the degradation profile of an ADC may reveal some limitations. For example, these assays do not measure whether the linker portion of the ADC is released with the drug attached despite potential for drug release from this complex. Similarly, circulating peptide fragments containing the linker/small-molecule complexes might be capable of releasing the drug. Without mass balance and profiling information, unmeasured small molecules may be present in a dose and cause differences in safety and efficacy despite similar PK profiles for the two lots.