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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.

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.

Peptide mapping with liquid chromatography–tandem mass spectrometry (LC-MS/MS) is an important analytical method for characterization of post-translational and chemical modifications in therapeutic proteins. Despite its importance, there is currently no consensus on the statistical analysis of the resulting data. In this manuscript, we distinguish three statistical goals for therapeutic protein characterization: (1) estimation of site occupancy of modifications in one condition, (2) detection of differential site occupancy between conditions, and (3) estimation of combined site occupancy across multiple modification sites. We propose an approach, which addresses these goals in terms of summarizing the quantitative information from the mass spectra, statistical modeling, and model-based analysis of LC-MS/MS data. We illustrate the approach using an LC-MS/MS experiment from an antibody-drug conjugate and its monoclonal antibody intermediate. The performance was compared to a ‘naïve’ data analysis approach, by using computer simulation, evaluation of differential site occupancy in positive and negative controls, and comparisons of estimated site occupancy with orthogonal experimental measurements of N-linked glycoforms and total oxidation. The results demonstrated the importance of replicated studies of protein characterization, and of appropriate statistical modeling, for reproducible, accurate and efficient site occupancy estimation and differential analysis.

Purpose Trastuzumab-DM1 (T-DM1) is an antibody-drug conjugate that uses trastuzumab to specifically deliver the maytansinoid antimicrotubule agent DM1 to HER2-positive cells. This first-in-human study of T-DM1 evaluated safety, pharmacokinetics, and preliminary activity of T-DM1 in patients with advanced HER2-positive breast cancer. Patients and Methods Successive cohorts of patients who had progressed on trastuzumab-based therapy received escalating doses of T-DM1. Outcomes were assessed by standard solid-tumor phase I methods. Results Twenty-four patients who had received a median of four prior chemotherapeutic agents for metastatic disease received T-DM1 at 0.3 mg/kg to 4.8 mg/kg on an every-3-weeks schedule. Transient thrombocytopenia was dose-limiting at 4.8 mg/kg; the maximum-tolerated dose (MTD) was 3.6 mg/kg. The half-life of T-DM1 at the MTD was 3.5 days, with peak DM1 levels < 10 ng/mL. Clearance at doses < 1.2 mg/kg was faster than at higher doses. Common drug-related adverse events (AEs) included grade ≤ 2 thrombocytopenia, elevated transaminases, fatigue, nausea, and anemia. No grade > 1 nausea, vomiting, alopecia, or neuropathy events and no cardiac effects requiring dose modification were reported. The clinical benefit rate (objective response plus stable disease at 6 months) among 15 patients treated at the MTD was 73%, including five objective responses. The confirmed response rate in patients with measurable disease at the MTD (n = 9) was 44%. Conclusion At the MTD of 3.6 mg/kg every 3 weeks, T-DM1 was associated with mild, reversible toxicity and substantial clinical activity in a heavily pretreated population. Phase II and III trials in patients with advanced HER2-positive breast cancer are under way.

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.

THIOMABs are antibodies with an engineered unpaired cysteine residue on each heavy chain that can be used as intermediates to generate antibody-drug conjugates. Multiple charge variant peaks were observed during cation-exchange chromatography (CEX) and imaged capillary isoelectric focusing (cIEF) analysis of several different THIOMABs. This charge heterogeneity was due to cysteinylation and/or glutathionylation at the engineered and unpaired cysteines through disulfide bonds formed during the cell culture process. Cysteine treatment followed by analysis using CEX, LC/MS and electrophoresis demonstrates that cysteine is a mild reductant that can remove glutathione and cysteine bound to the engineered cysteines without disrupting the inter- or intra-chain disulfide bonds of antibodies. We further demonstrated that using a cysteine/cystine redox pair (rather than cysteine alone) can not only effectively remove glutathione at the engineered cysteines, but also generate homogeneously cysteinylated species, which resulted in one main peak in both CEX-HPLC and imaged cIEF assays for antibodies with engineered and unpaired cysteines.

Protein aggregation is a common issue encountered during manufacture of biotherapeutics. It is possible to influence the amount of aggregate produced during the cell culture and purification process by carefully controlling the environment (eg, media components) and implementing appro-priate strategies to minimize the extent of aggregation. Steps to remove aggregates have been successfully used at a manufacturing scale. Care should be taken when developing a process to monitor the compatibility of the equipment and process with the protein to ensure that potential aggregation is minimized.