In the manufacture of the antibody-drug conjugate Trastuzumab-DM1 (T-DM1), the lysine residues on the antibody trastuzumab (Tmab) are modified to form the intermediate Tmab-MCC (T-MCC) and then conjugated with the drug DM1. Our goal is to understand the effects of modification and conjugation steps on the physicochemical stability of the antibody. The structural stability of Tmab relative to its modified and conjugated forms was assessed, employing thermally induced stress conditions to formulations containing Tmab, T-MCC, and T-DM1. DSC, SEC, CE-SDS, and LC-MS were used to study the stability of Tmab, T-MCC, and T-DM1 to thermal stress. The DSC thermograms show a decrease in melting temperature for the CH2 transition, in the order Tmab > T-MCC > T-DM1. As per SEC analysis, a significant increase in level of aggregation was detected in T-MCC (∼32%) and T-DM1 (∼5%) after 14 days at 40 °C. Tmab did not show significant aggregate formation. CE-SDS and LC-MS data demonstrate that the aggregation in the case of T-MCC is largely covalent and involves mechanisms other than formation of intermolecular disulfide cross-links. The aggregation observed for T-MCC was significantly inhibited upon addition of amino acids with nucleophilic side chains containing thiol (Cys) and hydroxyl moieties (Ser, Tyr). The covalent aggregation observed for T-MCC and the ability of nucleophilic amino acids, particularly Cys, to inhibit it indicate that the maleimide moiety in the MCC linker may react to form intermolecular covalent cross-links between T-MCC molecules, possibly through a Michael addition mechanism. In addition, DSC results demonstrate that the conjugation of the drug moiety DM1 to Tmab results in destabilization of the CH2 domain of the antibody.

Trastuzumab emtansine (Kadcyla) is a recently approved antibody-drug conjugate produced by attachment of the anti-tubulin drug, DM1, to lysine amines via the SMCC linker. The resulting product exhibits a drug load distribution from 0 to 8 drugs per antibody that can be quantified using mass spectrometry. Different statistical models were tested against the experimental data derived from samples produced during process characterization studies to determine best fit. The Poisson distribution gives the best correlation for samples manufactured using the target process conditions (yielding the target average drug to antibody ratio (DAR) of 3.5) as well as those produced under conditions that exceed the allowed manufacturing ranges and yield products with average DAR values that are significantly different from the target (i.e., ≤3.0 or ≥4.0). The Poisson distribution establishes a link between average DAR values and drug load distributions, implying that measurement and control of the former (i.e., via a simple UV spectrophotometric method) could be used to indirectly control the latter in trastuzumab emtansine.

The antibody-drug conjugate, trastuzumab emtansine (Kadcyla), is produced by attachment of the antitubulin drug, DM1, to lysine amines via a heterobifunctional linker, SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). Following the reaction of the N-hydroxysuccinimide activated linker with antibody lysines to produce a linker-modified intermediate (Tmab-MCC), DM1 is added to yield the desired product. In addition to the expected distribution of drug-linked forms (from 0 to 8), mass spectrometry also demonstrates the presence of a second distribution shifted by about +222 Da. This series is consistent with the presence of a population containing a bound linker without DM1 ("unconjugated linker"). Extended characterization of trastuzumab emtansine was performed using capillary isoelectic focusing, CE-SDS, peptide mapping, and LC/MS following (18)O labeling of peptide digests to identify this family of product variants. These studies demonstrate that the presence of these +222 Da species is due to an unexpected reaction of the maleimide moiety in the MCC linker with antibody lysine residues to produce cross-linked species that cannot conjugate to DM1.

This study describes the use of a multidimensional HPLC (2D and 4D) system for a faster and more effective characterization of an antibody–drug conjugate (ADC) product, compared to the standard off-line approach of fraction collection and off-line variant characterization. The size variants of an interchain cysteine-linked ADC were characterized to understand the effect of the different drug-to-antibody ratio (DAR) species on aggregate formation. For this purpose, the ADC product and a full panel of stressed samples were analyzed. The dimeric ADC species were baseline resolved from the main peak (Rs = 2.7) by UHP-SEC (ultra-high-performance size exclusion chromatography) under nondenaturing conditions using a buffered mobile phase containing 5% 2-propanol. A 2D-LC (SEC-HIC) method was then developed to compare the average DAR values of the main peak species vs the aggregates. A 4D-LC/MS method (SEC-reduction-digestion-RPHPLC) was also developed to determine levels of potential critical quality attributes (pCQAs) including aggregation, average DAR, oxidation, and deamidation, in a 2 h run. An average DAR value of 3.5–3.6 was found for the main peak using both 2D-LC and 4D-LC methods, and these values were consistent with DAR determined by the in-house reference hydrophobic interaction chromatography (HIC) method. The multidimensional LC approaches also showed an increase in the content of high-DAR species in the SEC fractions containing the aggregates. Overall the entire workflow of data acquisition is completed within a day using the multidimensional on-line approach, in comparison to multiple days required with the traditional off-line approaches.

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