Molecular recogntion assays of critical structure attributes in proteoforms

A luminon assay method for analyzing a critical structure attribute in a proteoform or proteoform family through sequestration of one or more affinity selectors, comprising the steps of:

• a) chronologically selecting a cell and particulate-clarified sample from a process development or production fermenter, a cell culture, or organism,
• b) continuously adding mobile phase bearing a fluorescent low molecular weight labeled molecular recognition agent (Mra) to a size exclusion chromatography (SEC) column, the Mra being either a primary (Pas) or secondary (*Sas) affinity selector,
• c) injecting a sample into the SEC column, wherein;
• (i) the linear velocity of the analyte protein (Ap) is more than twice the size of the Mra,
• (ii) the greater linear velocity of the Ap relative to the Mra causes the two to mix, thereby forming a luminon complex (Ap:Mra), and
• (iii) the high linear velocity of the Ap:Mra complex causes the complex to move into a saturating concentration of Mra and be washed repeatedly as it migrates to a flow-through detector,
• d) transporting the sequestome complex thus resolved into a flow-through detection means;
• and
• e) using the flow-through detection means, detecting and quantifying critical structure attributes (CSAs) in analytes sequestered within the sequestome complex.

A method for in-line monitoring of one or more critical structure attributes (CSAs) in a proteoform or proteoform family by analyte sequestering of affinity selectors, comprising the steps of:

• a) continuously providing a cell and particulate clarified sample stream, including the proteoform or proteoform family, from a fermenter, cell culture, or organism;
• b) continuously adding affinity selector reagents to the sample stream to form a mixture, wherein
• (i) at least a first reagent is labeled with a donor fluorophore (**Pas) that binds specifically to a protein analyte, and
• (ii) at least a second reagent is labeled with an acceptor fluorophore (*Sas) that binds to a critical structure attribute in the protein analyte;
• c) introducing the mixture of reagents and sample continuously through a stationary micro-mixer into an incubation or SEC column, wherein;
• (i) reagents concentration exceeds anticipated levels of the analyte and CSAs, and
• (ii) the combined volume of reagents is less than that of the sample;
• d) adapting the flow rate through the reactor or SEC column to allow formation of one or more fluorescence labeled (**Pas)m:An:(*Sas)n complexes;
• e) transporting the complexes to a fluorescence detection means capable of detecting a single or multiple analytes, wherein
• (i) the detection means includes Förster resonance energy transfer (FRET) spectroscopy, or
• (ii) detection is achieved by a change in fluorescence after association of the affinity selectors with the analyte;
• f) identifying and quantifying specific critical structure attributes (SCAs) of the proteoform or proteoform family based on selector fluorophore spectral properties for use in process validation.