A framework for calculating orthogonal selectivities in multimodal systems directly from cell culture fluid

Area of Science:

• Biotechnology
• Analytical Chemistry
• Protein Separation Science

Background:

• Selecting optimal chromatography resins is crucial for efficient biopharmaceutical purification.
• Quantifying resin orthogonality in complex mixtures like cell culture fluids is challenging.
• Existing methods often require laborious protein tracking.

Purpose of the Study:

• To develop a direct and efficient method for measuring orthogonality between multimodal chromatography resins.
• To enable rational selection of resins and operating conditions for host cell protein (HCP) removal.
• To facilitate the development of next-generation multimodal resins tailored for specific expression systems.

Main Methods:

• Fractionation of null-producing cell culture fluids (CCFs) using multimodal resins with linear salt gradients.
• Analysis of fractions by ultra-performance reversed-phase liquid chromatography (UP-RP-LC) to generate HCP "fingerprints."
• Application of an inner product vector-based approach to quantify orthogonality between resins and conditions.

Main Results:

• Orthogonality is highly dependent on the expression system (e.g., Chinese hamster ovary, Pichia pastoris) and specific HCPs.
• Unexpectedly orthogonal resin pairs within the same family were identified.
• Optimal operating regimes for maximizing orthogonal selectivity were determined, including "self-orthogonality" at different pHs.

Conclusions:

• The developed framework provides a labor-saving method to quantify resin orthogonality without protein tracking.
• This approach significantly aids in process development and resin selection for monoclonal antibody (mAb) and non-mAb purification.
• The study unlocks the potential of multimodal resins by enabling their rational design and selection for diverse bioprocessing needs.