Statistics of the distribution of the abundance of molecules with various drug loads in maytansinoid antibody-drug conjugates.

The maytansinoid antibody-drug conjugates (ADCs) in clinical development for cancer therapy each contain a derivative of the microtubule-targeting agent, maytansine, covalently attached to the antibody via an engineered linker. A sample of any of these conjugates contains molecules with different numbers of maytansinoid molecules, or "drug" loads, the relative abundance of which can be determined by mass spectrometry. We examined the accuracy of the Poisson distribution and the binomial distribution in predicting the relative abundance of species with different drug loads for three antibody-maytansinoid conjugates with different antibodies and linker-maytansinoid pairings. We used variance, calculated from the experimental mass distribution data, as the parameter to determine the optimal value n of the binomial distribution number of trials. The accuracy of the Poisson distribution in predicting distribution of the species abundance in these conjugates varied among the conjugates. In contrast, the accuracy of the binomial distribution was similar for all three conjugates and comparable to the best accuracy of the Poisson distribution, as supported by a paired t-test.

Structural characterization of the maytansinoid-monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry.

Immunoconjugates are being explored as novel cancer therapies with the promise of target-specific drug delivery. The immunoconjugate, huN901-DM1, composed of the humanized monoclonal IgG1 antibody, huN901, and the maytansinoid drug, DM1, is being tested in clinical trials to treat small cell lung carcinoma (SCLC). huN901-DM1 contains an average of three to four DM1 drug molecules per huN901 antibody molecule. The drug molecules are linked to huN901 through random modification of huN901 at epsilon-amino groups of lysine residues, thus yielding a heterogeneous population of conjugate species. We studied the drug distribution profile of huN901-DM1 by electrospray time-of-flight mass spectrometry(ESI-TOFMS), which showed that one to six DM1 drug molecules were attached to an antibody molecule. Both light and heavy chains contained linked drugs. The conjugation sites in both chains were determined by peptide mapping using trypsin and Asp-N protease digestion. Trypsin digestion identified modified lysine residues, since these residues were no longer susceptible to enzymatic cleavage after conjugation with the drug. With respect to Asp-N digestion, modified peptides were identified by observing a mass increase corresponding to the modification. The two digestion methods provided consistent results, leading to the identification of 20 modified lysine residues in both light and heavy chains. Each lysine residue was only partially modified. No conjugation sites were found in complementarity determining regions (CDRs). Using structural models of human IgG1, it was found that modified lysine residues were on the surface in areas of structural flexibility and had large solvent accessibility.

Structural characterization of a recombinant monoclonal antibody by electrospray time-of-flight mass spectrometry.

PURPOSE: The aim of this study was to perform structural characterization of a recombinant monoclonal antibody (MAb), huN901, by electrospray time-of-flight mass spectrometry (ESI-TOFMS) using both "top-down" and "bottom-up" approaches. METHODS: In the top-down approach, the molecular masses of the deglycosylated huN901 and the light and heavy chains of the antibody were measured by direct infusion MS and liquid chromatography-mass spectrometry (LC-MS). In the bottom-up approach, trypsin and Asp-N protease were used to digest the separated, reduced and alkylated light and heavy chains followed by LC-MS analysis of the digests. RESULTS: The primary structure and post-translational modifications of huN901 were characterized by both top-down and bottom-up MS approaches. Modifications of N-terminal pyroglutamate formation, cleavage of C-terminal lysine, glycosylation, and deamidation were identified in the antibody heavy chain by both protein mass measurement and peptide mapping. No modifications were found in the complementarity determining regions (CDRs) of both chains. Both trypsin and Asp-N protease digestion had an average sequence recovery of 97%, and generated complimentary mapping results with complete sequence recovery. CONCLUSIONS: ESI-TOFMS is a superior tool to characterize MAb and other complex protein pharmaceuticals.

Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometry.

Recombinant monoclonal antibody drug products play an increasingly important role in the treatment of various diseases. Antibodies are large, multi-chain proteins and antibody preparations often contain several molecular variants, which renders them heterogeneous. The heterogeneity is further increased in immunoconjugates prepared by covalently linking several drug molecules per antibody molecule. As part of the product characterization, the molecular weights of the antibodies or their drug conjugates need to be measured. Electrospray ionization mass spectrometry (ESI-MS) is well suited for the analysis of recombinant antibodies and immunoconjugates. Sample preparation is an important element of ESI-MS analysis, in particular samples need to be freed of interfering charged species, such as salts and buffer components. In this paper, Amicon centrifugal filters, reversed-phase high-performance liquid chromatography (HPLC), and size-exclusion HPLC were evaluated for sample desalting. Size-exclusion HPLC, using aqueous acetonitrile as the mobile phase, directly coupled to ESI-MS provided the best performance and was optimized for the study of immunoconjugates. The results showed that antibodies carrying covalently linked maytansinoid molecules generated charge envelope profiles that differ from those of the non-conjugated antibody. For the determination of the distribution of the various conjugate species in an immunoconjugate sample prepared by randomly linking in the average 3.6 drug molecules per antibody molecule, the experimental conditions needed to be carefully selected to allow acquisition of the whole spectrum containing the charge envelopes of all species.

A reversed-phase high-performance liquid chromatography method for analysis of monoclonal antibody-maytansinoid immunoconjugates.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for detection and quantification of free maytansinoid drug in disulfide-linked conjugates between monoclonal antibodies and the maytansinoid drug DM1 (MAb-DM1). Mobile phases and gradient conditions were optimized for separation of several DM1-related free drug species from MAb-DM1 conjugates. The selectivity, linearity, and reproducibility of the method are reported. Reduction of the disulfide-linked DM1 followed by RP-HPLC allowed estimation of purity of MAb-linked DM1 as well as recovery of L-DM1. The method was also used to estimate drug per MAb ratios, which were consistent with those determined by UV spectroscopy.