Evaluating the Bioequivalence of Antibody–Drug Conjugates - Pharmaceutical Technology

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Evaluating the Bioequivalence of Antibody–Drug Conjugates
The authors discuss the analytical methods and related testing for bioequivalence studies of ADCs. This article is part of a special issue on analytical technology.

Pharmaceutical Technology
pp. s22-s27

Test article

ADCs are composed of three distinct portions: the mAb, linker, and cytotoxin, each of which has unique properties from a manufacturing and characterization perspective. For example, the mAb portion of Mylotarg was produced in a mammalian-cell suspension culture using a cell line and was purified using three independent steps (i.e., low pH treatment, diethylaminoethyl (DEAE) Sepharose chromatography, and viral filtration) to achieve antibody purification with acceptable retrovirus inactivation and removal (10). Mylotarg release testing included SDS–PAGE, IEF, HPLC, ELISA, aggregate and unconjugated calicheamicin ozogamicin, endotoxins by limulus amebocyte lysate and bioburden, protein content, peptide mapping, and oligosaccharide profiling (9). Potency was tested using a cytotoxicity assay and an immunoaffinity antigen-binding ELISA (9).

Figure 2: Conjugation-related antibody drug conjugate variants using hydrophilic interaction liquid chromatography: MR0 is unbound free monoclonal antibody (mAb), MR1 is one cytotoxin, MR2 are two cytotoxins, MR3 are three cytotoxins, MR4 are four cytotoxins, MR6 are six cytotoxins, and MR 8 are eight cytotoxins. Reproduced with permission from Seattle Genetics from K. Anderson, "Physiochemical Characterization of Aurustatin-based Antibody Drug Conjugates," presented at the National Biotechnology Conference (San Francisco, 2011). (FIGURE 2 IS COURTESY OF SEATTLE GENETICS AS CITED, RESPECTIVELY, WITH CREDIT IN FIGURE 2.)
The characterization of a manufactured lot of an ADC usually reveals the amount of free and bound mAb protein as well as the mAb-to-drug ratio. Cytotoxic agents, such as DM1, have an absorption maximum at 252 nm, and mAbs absorb at 280 nm. Therefore, the amount of drug bound to mAb can be determined using differential-absorption measurements at 252 nm and 280 nm (17). Typical ADC drug-to-mAb ratios are between 2 and 4, as seen in hydrophilic interaction liquid chromatography (HILC) analysis of ADCs (see Figure 2) and reverse-phase chromatographic analysis of ADCs (see Figure 3) (18, 19).

Figure 3: The drug load of antibody–drug conjugates can be determined by reverse-phase chromatography. Reproduced with permission from Seattle Genetics, from R.P. Lyon, et al., "Development of Parallel Conjugation and Assay Methodologies to Screen for Antibodies with Optimal Properties for use as Antibody-Drug Conjugates," presented at the 101st Annual American Association for Cancer Research Meeting (Washington DC, 2010). (FIGURE 3 IS COURTESY OF SEATTLE GENETICS AS CITED, RESPECTIVELY, WITH CREDIT IN FIGURES 3.)
This gross characterization of each lot is problematic for several reasons. First, the nonconjugated antibody (i.e., free antibody) can bind with the same or even better avidity as the ADC, but the contribution of this component to product efficacy or toxicity is not systematically evaluated during ADC drug development. Preclinical efficacy studies should include a quantitative assessment of the impact of the free antibody in the mixture by using multiple manufacturing lots to capture the effect of that variability.

Second, the average drug-load-per-antibody value does not ensure the efficacy of a particular lot. For example, an antibody ratio of four molecules of cytotoxin per antibody could result if all ADCs in the lot have four drug molecules attached, if 50% of the antibodies have eight drug molecules attached and 50% were free antibody, or if 25% of the antibodies have drug attached and 75% are drug-free antibodies. In the latter two examples, the free antibody would compete for binding with the conjugated antibody, thus limiting that particular lot's efficacy. Knowing the quantitative distribution of conjugation ratios is critical for batch-release testing. Similarly, the location of the conjugation reaction can affect activity, and in vivo liability should be evaluated. Finally, understanding the rate of in vivo cleavage at each conjugation site allows modeling of the outcome of a particular manufacturing lot if quantitative assessment of the species distribution is available.

The manufacturing process to link the small molecule to the antibody usually favors covalent binding to the hydrophilic domains of the antibody through linking to the sulfhydryl (SH) group on cysteine residues or coupling to the amine (NH2) group on lysine molecules (18, 19). Thermodynamically, most conjugation reactions occur at the most accessible and lowest energy-barrier binding sites. While an in vitro efficacy assay can assess the potency of an individual manufacturing lot at the time of release, the dynamic loss of the linker-small-molecule complex that occurs upon dosing can cause different outcomes in vivo. In vivo exposure to the aqueous environment and endogenous enzymes could cause these easily accessible sites to be prone to degradation. Therefore, ADCs with only one or two small molecules attached may be more easily converted to the free mAb and block ADC interactions with the target molecule. Scientist still have insufficient data to predict this site-specific degradation or even the degradation dynamics in vivo.


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