PEGS Boston was wonderful. We met many new people and companies, and MAM is increasingly adopted. In five days of conversation with antibody discovery scientists, CMC analytical teams, biopharma proteomics groups and CROs, several topics were common.

Many discussions were about where and how MAM assists antibody and biologics characterization, how it integrates with existing release and stability assays, what it takes to qualify a MAM workflow and how a standardized workflow can reduce method development from fermentation to formulation. People were very interested in the possibility of using one standard, unchanging kit at every stage.

An assay designed to measure a particular attribute (e.g. deamidation, oxidation, isomerization, clipping…) must not introduce or alter the very modifications being measured. People were interested in the ProtiFi™ S-Trap™ MAM Kit because its rapid, on-trap digestion does not induce such changes. MAM kit processing of the NIST monoclonal antibody RM 8671 reduced deamidation at the N388 hotspot in the PENNY peptide (residues 375 to 396) >99% compared with conventional in-solution procedures. Accurate QC requires this level of sample fidelity.

Concerns about contaminant removal efficiency were common. Biologic drug formulations are highly variable and can contain salts, excipients, detergents, polymers, stabilizers and surfactants like polysorbates (PS). Indeed, PS20 and PS80 are in >80% of commercial monoclonal antibody formulations and are demonstrated to remain after published MAM workflows. Such contaminants interfere with downstream chromatography and MS analysis, degrading sensitivity especially identification and quantification of host cell proteins (HCPs), and new peak detection.

We presented our results on testing 41 foulants found in biologic formulations including detergents, polymers, salts, surfactants, and potential process contaminants present from fermentation through formulation. The MAM kit fully removed all tested contaminants without change in protocol. It is suited to immediate use across a wide range of product types and process stages.

Discussions also touched on digestion strategies. In comparison to the published NIBRT MAM workflow, the kit delivered >10x antibody signal, lower background levels, >35× more HCP peptide IDs and 3x the number of HCPs. Additionally, applying Tryp-N™, or “mirror trypsin”, cleaves before K/R, rather than after them to generate b-ion-rich spectra that complement trypsin, yielded 100% sequence coverage of both light and heavy chains of NIST mAb 8671. As “mirror trypsin”, Tryp-N™ cleaves before K/R, rather than after, and generates b-ion-rich spectra that complement trypsin; this enables confident localization of PTMs and single-amino-acid variants, and controlled partial digestion fills sequence gaps.

MAM is used and is being applied to many new biologics and formulations. Sample preparation must deliver stable results that faithfully observe the actual state of the biologic under study and do not introduce artifactual chemical changes. New digestion strategies yield full sequence coverage and localization of PTMs. The kit workflow minimizes prep-induced modifications and removes a very large variety of formulation contaminants; this allows standardization of MAM across discovery, development and QC environments.

We will be at ASMS in San Diego from May 31 through June 4. Come visit to see the full ProtiFi™ workflow lineup, including the S-Trap™ MAM Kit, S-Trap™ Turbo™, Si-Trap™, Tryp-N™, and SimpliFi™. If MAM, peptide mapping, contaminant removal, or orthogonal digestion are of interest, swing by!