Fred Regnier

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 …

A system and method is provided for validating the manufacturing process for the production of complex biological compositions, and particularly for providing process validation information for evaluation by a federal regulatory agency. The system and method continuously and chronologically assess the concentration of proteoforms within the biological composition as it is being produced in a fermentor. Samples from the fermentor are analyzed in a pre-selected array of analysis columns, with data generated by the columns being accumulated and evaluated, and particularly compared with data from previous stages in the production process. A continuous process validation system includes top-down and bottom-up analysis sectors, each including a plurality of different analysis columns that can be selected by the controller for a particular biological composition and a particular production process.

Proteomes may be composed of a million or more protein species. Finding and quantifying a single protein in samples of this complexity is a great challenge for liquid chromatography-mass spectrometry (LC-MS) systems. This problem is complicated by the fact that proteomes bear proteoform families that vary slightly in primary, secondary, or tertiary structure and posttranslational modifications. Moreover, members of these families often vary in biological activity. The fact that many proteoforms are not in sequence libraries is a further problem. All of these variables complicate the interpretation of LC-MS data. A problem with LC-MS systems is that irrespective of their resolving power they lack the ability to select molecular species for analysis on a structural basis. It would be of great value to group polypeptides for analysis according to structural features instead of identifying those features after an analysis is …