Monoclonal antibodies (mAbs) are the fastest growing class of therapeutic drugs, because of their high specificities to target cells. Facile analysis of therapeutic mAbs and their post-translational modifications (PTMs) is essential for quality control, and mass spectrometry (MS) is the most powerful tool for antibody characterization. This study uses pepsin-containing nylon membranes as controlled proteolysis reactors for mAb digestion prior to ultrahigh-resolution Orbitrap MS analysis. Variation of the residence times (from 3 ms to 3 s) of antibody solutions in the membranes yields “bottom-up” (1–2 kDa) to “middle-down” (5–15 kDa) peptide sizes within less than 10 min. These peptides cover the entire sequences of Trastuzumab and a Waters antibody, and a proteolytic peptide comprised of 140 amino acids from the Waters antibody contains all three complementarity determining regions on the light chain. This work compares the performance of “bottom-up” (in-solution tryptic digestion), “top-down” (intact protein fragmentation), and “middle-down” (in-membrane digestion) analysis of an antibody light chain. Data from tandem MS show 99%, 55%, and 99% bond cleavage for “bottom-up”, “top-down”, and “middle-down” analyses, respectively. In-membrane digestion also facilitates detection of PTMs such as oxidation, deamidation, N-terminal pyroglutamic acid formation, and glycosylation. Compared to “bottom-up” and “top-down” approaches for antibody characterization, in-membrane digestion uses minimal sample preparation time, and this technique also yields high peptide and sequence coverage for the identification of PTMs.

Proteolytic digestion is an important step in characterizing protein sequences and post-translational modifications (PTMs) using mass spectrometry (MS). This study uses pepsin- or trypsin-containing spin membranes for rapid digestion of single proteins or simple protein mixtures prior to ultrahigh-resolution Orbitrap MS analysis. Centrifugation of 100 μL of pretreated protein solutions through the functionalized membranes requires less than 1 min and conveniently digests proteins into large peptides that aid in confirming specific protein sequence variations and PTMs. Peptic and tryptic peptides from spin digestion of apomyoglobin and four commercial monoclonal antibodies (mAbs) typically cover 100% of the protein sequences in direct infusion MS analysis. Increasing the spin rate leads to a higher fraction of large peptic peptides for apomyoglobin, and MS analysis of peptic and tryptic peptides reveals mAb PTMs such as N-terminal pyroglutamate formation, C-terminal lysine clipping and glycosylation. Relative to overnight in-solution digestion of mAbs, spin digestion yields higher sequence coverages. Spin-membrane digestion followed by infusion MS readily differentiates a mAb to the Ebola virus from a related antibody that differs by addition of a single amino acid.

Establishing stability at all stages of a sample’s lifespan is a critical part of performing regulated bioanalysis. For plasma assays, this includes the duration between when blood is drawn and when that blood is centrifuged to produce plasma. Here, we provide a discussion of current regulatory expectations around whole blood stability testing for LC–MS plasma assays, as well as the two primary experimental approaches utilized to assess whole blood stability. Next, we interrogated a large dataset of validated methods (1076 methods, the vast majority of which were for measurement of small molecules) to assess the correlation between whole blood and plasma stability profiles, finding them to be highly correlated. Finally, we summarize unique case studies; we have encountered during WB stability testing which offer lessons that may be broadly applicable.

Aim: To further enhance the detection sensitivity and increase resolving power of top-down intact protein bioanalysis, middle-down approach was explored. Materials & methods: An monoclonal antibody (mAb) was used as a model protein to evaluate quantitative bioanalytical assay performance and a disulfide linked dimer protein was investigated for its pharmacokinetics properties and catabolism in vivo by middle-down approach. Results & Conclusion: For quantitation of the mAb, different subunits generated by middle-down approach provided different level of signal improvement in biological samples with Lc and half Fc giving five-times better sensitivity than intact mAb. For the dimer protein, middle-down analysis by reduction enabled effective differentiation of the unchanged protein and its oxidized form, and clearly elucidated their respective proteolytic catabolites.

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