Analytical methods for bioequivalence studies
Analytical-method development for biologics usually starts with establishing a suitable manufacturing process, followed by
the development of analytical methods intended to confirm adequate structural and functional comparison with the target product
profile (i.e., for ADCs, the starting mAb). The regulatory guidelines about this development require an understanding of the
molecular structure, glycol microheterogeneity profile, impurities, degradation products, and bioavailability, as shown in
Table I (13–16).
Table I. Analytical technologies for assessment of biologics.
The normal complexity in evaluating lot-to-lot variation and its implications is further compounded because each of the three
major components (mAb, linker, and cytotoxin) must be fully characterized. According to EMA's biosimilar guidelines, "Every
mAb is unique and small structural changes can have significant functional consequences since even the same expression system
and similar culture conditions might lead to a distinct product profile (e.g., impurities and microheterogeneity)" (14). Manufacturing
modifications or synthetic-process changes may require further nonclinical and clinical studies.
The structural integrity of ADCs is evaluated using techniques such as amino acid sequencing (N- or C- terminal), biochemical testing, carbohydrate mapping, high-performance liquid chromatography (HPLC), electrophoresis (i.e.,
sodium dodecyl sulfate polyacrylamide gel electrophoresis [SDS-PAGE], isoelectric focusing [IEF], and Western blotting), immunoassays
(enzyme-linked immunosorbent assay [ELISA] and Gyros), macromolecular liquid chromatography–mass spectrometry (MS)/MS (LC–MS/MS),
nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopy, X-ray crystallography, peptide mapping, ultracentrifugation,
and other physiochemical methods to show that the purified mAb is not fragmented, aggregated, or otherwise modified. Specificity
characterization can be performed using in vitro cell-based assays or in vivo animal models and should provide evidence that the ADC is specific for its intended target and has low cross-reactivity with
human tissues. Potency characterization is used for assessment of lot-to-lot consistency and stability and may involve techniques,
such as ELISA, Gyros, flow cytometry, cytotoxicity, and animal models.
Most mAbs are produced in bioreactors, so DNA, RNA, viral burden, host-cell protein, endotoxins/pyrogenicity, and growth-media
components all must be monitored and controlled in the final ADC product. mAbs are glycosylated proteins composed of four
peptide chains connected by disulfide bridges. The oligosaccharide residues on mAbs may be involved in activity, and, therefore,
mAbs should be characterized for protein, peptide sequence, glycoprotein, and oligosaccharide content. The binding activity,
affinity, avidity, immunoreactivity, source of all materials, chemical structures and production processes, purity, viral
load, and the average mAb-to-drug ratio all must be clearly defined. Contaminants and impurities must be carefully controlled,
monitored, and minimized. Stability should be carefully monitored for intact ADC and bioactivity. Immunogenicity is a major
concern for nonhumanized mAbs and must be thoroughly evaluated (16). Bioavailability, receptor binding, stability, and immunogenicity
are major issues of pharmaceutical equivalence for ADCs.