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).
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 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
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
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.)
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.