An integrated analytical process for chronologically assessing the concentration of proteoforms in a family of protein variants contained within complex biological compositions obtained from a fermentor that is part of a manufacturing process for the production of the complex biological compositions, the compositions derived from an expanding native or genetically engineered cell culture of microbial, plant, animal, or human origin, the process comprising the steps of:
- a) providing at least a two-sector separation system including;
- a top-down (TD) sector including a plurality of distinct TD analysis columns that are configured and operable for intact proteoform resolution, detection of specific analyte proteoforms in samples bearing host-cell proteins, proteoforms, and metabolites, identifying and quantifying these components for the purpose of chronologically validating manufacturing process reproducibility;
- a bottom-up (BU) sector including a plurality of distinct BU analysis columns configured and operable for affinity purification and proteolysis of proteoforms during transport into the BU sector;
- and
- a controller configured and operable to select and assemble analytical protocols that will identify signature peptides while the two sectors are operating independently, either in tandem or parallel;
- (b) choosing conditions, sampling times, analytical method steps, and a system configuration for the TD sector to automate the generation of analytical data to validate a manufacturing process incorporating the fermentor, wherein data from individual analytical steps is chronologically converted by the controller into digital data blocks that provide a means to evaluate i) analyte variation during a production run relative to earlier time points within the process and equivalent chronological steps in prior production runs, ii) the presence of proteoforms bearing positive and negative structural attributes that impact human health, iii) the need for short term and long term remediation of process deviations, and iv) process continuity relative to prior production runs,
- (c) obtaining analytical samples of the complex biological composition from the fermentor at designated times and removing particulate matter prior to delivery of the analytical sample to one of the sectors;
- (d) maximizing proteoform resolution of a specific monoclonal antibody proteoform family in multiple chromatographic or electrophoretic separation modes of differing selectivity, within one or more of the TD and BU analysis columns, to identify and quantify proteoforms by separation methods alone;
- (e) detecting individual members of an analyte family in the presence of host-cell proteins as they elute from analysis columns in the TD sector;
- (f) sequentially accumulating and time stamping analyte detection data from samples within a proteoform family passing through the TD sector, and acquiring contemporaneous data from sensors detecting the environment of the fermentor, instrument performance data, and run-time data, and then constructing time-versus-analog response curves that are digitized and saved as digital data by the controller,
- (g) comparing current data with data from earlier sample times and samples and chronologically equivalent data from prior production runs;
- whereupon qualitative and quantitative similarities between the current and earlier data is used by the controller to assess one or more potential deviations in the manufacturing process;
- (h) confirming the one or more potential deviations in the BU sector using either a proteoform derived from the TD sector or an unfractionated proteoform family that has been affinity selected, reduced or digested with a Pep-Fc-proteolysis column before analysis of the resulting peptide classes in the BU sector, wherein the affinity column is resistant to trypsin digestion;
- (i) providing individual analyses of the peptide classes by one of multiple selective modes of liquid chromatography designated by the controller in combination with mass spectrometry, wherein the liquid chromatography modes can be by phenylboronate, reversed phase, hydrophilic interaction, or molecular recognition-based affinity chromatography;
- (j) providing absorbance and/or mass spectral detection and analysis of the separated signature peptides in the BU sector;
- (k) providing quantification of the peptides through multiple reaction monitoring by mass spectrometry;
- (l) chronologically converting analytical data from one or more detectors in the BU sector into digital data that can be evaluated by the controller to assess and confirm i) variations during a production run relative to earlier time points within the process and equivalent chronological steps in prior production runs, ii) the presence of signature proteoforms bearing positive and negative structural attributes that impact human health, iii) the need for short term and long term remediation of process deviations, and iv) process continuity relative to prior production runs, and
- (m) constructing a digital provenance of the manufacturing record at the end the production run based on the chronological analytic data that can be evaluated by a federal regulatory agency and that can be subsequently used to improve the process.