Novilytic (United States)

Federal regulatory agencies require continuous verification of recombinant therapeutic monoclonal antibody (mAb) quality that is commonly achieved in a two-step process. First, the host-cell proteome and metabolome are removed from the production medium by protein A affinity chromatography. Second, following recovery from the affinity column with an acidic wash, mAb quality is assessed in multiple ways by liquid chromatography-mass spectrometry (LC-MS). However, lengthy sample preparation and the lack of higher-order structure analyses are limitations of this approach. To address these issues, this report presents an integrated approach for the analysis of two critical quality attributes of mAbs, namely titer and relative aggregate content. Integration of sample preparation and molecular-recognition-based analyses were achieved in a single step utilizing an isocratically eluted mobile affinity selection chromatography (MASC) column. MASC circumvents the protein A step, simplifying sample preparation. Within 10 min, (i) mAbs are fluorescently coded for specific detection, (ii) monomers and aggregates are resolved, (iii) the mAb titer is quantified, (iv) relative aggregate content is determined, (v) analytes are detected, and (vi) the column is ready for the next sample. It is suggested herein that this mode of rapid quality assessment will be of value at all stages of discovery (screening, clone selection, characterization), process R&D, and manufacturing. Rapid monitoring of variant formation is a critical element of quality evaluation.

This chapter focuses on analytical and chemical approaches to the study of oxidative stress based on a rationale and set of facts many laboratories have used for the past decade. Oxidative stress is closely associated with a series of diseases, producing carbonylated proteoforms specific biomarkers. Oxidative stress-based posttranslational modifications (PTMs) monitoring will become common in clinical diagnostics within the next decade. Protein carbonylation stems from four types of PTMs: polypeptide backbone cleavage, oxidization of amino acid side chains, nucleophilic addition of carbonyl containing metabolites from lipid oxidation, and oxidation products of glycation. The chapter discusses these four modification pathways. The purpose of carbonyl derivatization is to aid in oxidized protein purification, isotope labeling, and/or detection. A major part of current strategies for studying oxidized proteins is the use of affinity methods that recognize carbonylation sites. Oxidation of amino acids in proteins can also occur without the formation of carbonyl groups.