Pre-Capture Flocculation for Protein A Antibody Purification
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Solution Overview
Problem
The high cost and limited lifetime of affinity chromatography resins, particularly Protein A resins, and the issue of resin fouling and precipitations during low pH elution in the purification of monoclonal antibodies, necessitate a more efficient and cost-effective purification method.
Innovation Solution
Incorporating a flocculation step followed by depth filtration and affinity chromatography, using cationic compounds like poly(diallyldimethylammonium chloride) to agglomerate impurities, reducing the burden on subsequent capture chromatography steps, and employing additional polishing steps to enhance purity and resin reuse.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If affinity chromatography with Protein A is used for capture step, then selectivity for Fc-bearing molecules is improved (removing more than 99.5% of contaminants), but cost of purification increases significantly (resin is approximately 30 times more expensive than ion exchange resin) and resin lifetime is limited due to fouling
Solution Approach 1:
The patent applies preliminary clarification steps (depth filtration, centrifugation) before affinity chromatography to remove particles and aggregates from crude cell culture fluids. This pre-treatment prevents resin fouling during the affinity capture step, extending resin lifetime and reducing replacement frequency, thereby lowering overall process costs while maintaining high purification selectivity
Solution Approach 2:
The patent segments the purification process into distinct stages: (1) preliminary clarification to remove particles, (2) affinity capture for high-selectivity purification, and (3) polishing steps for final purification. This segmentation allows each step to perform its specific function optimally, preventing contamination accumulation on the expensive affinity resin and reducing its replacement frequency
2Device complexity
If capture steps are performed with crude input materials, then process complexity is reduced, but resin fouling increases leading to precipitations and reduced resin lifetime
Solution Approach 1:
The patent implements preliminary clarification steps (depth filtration, centrifugation) before affinity chromatography to remove particles and aggregates from crude cell culture fluids. This pre-treatment prevents resin fouling during the affinity capture step, extending resin lifetime and reducing replacement frequency, thereby lowering overall process costs while maintaining high purification selectivity
Solution Approach 2:
The patent segments the purification process into distinct stages: (1) preliminary clarification to remove particles, (2) affinity capture for high-selectivity purification, and (3) polishing steps for final purification. This segmentation allows each step to perform its specific function optimally, preventing contamination accumulation on the expensive affinity resin and reducing its replacement frequency
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method significantly reduces the overall purification costs and extends the lifetime of chromatography materials while maintaining high purity and yield of monoclonal antibodies, addressing the challenges of resin fouling and precipitations.
Implementation Method 1
adding a flocculation-inducing compound to cell culture fluid
Implementation Method 2
depth filtration of the mixture
Implementation Method 3
exposing the filtrate obtained in step (b) to affinity chromatography, wherein the immunoglobulin is bound to the affinity chromatography medium
Data Source
AI summary
The present invention relates to new methods for the purification of immunoglobulins from cell culture harvest. These methods are characterized by cleaning steps preceding an initial capture chromatography step. Preferably, the initial capture chromatography is an affinity chromatography and the pre-capture cleaning effect is obtained by flocculation and filtration. By using such pre-capture cleaning steps, an improved quality of the eluted immunoglobulin from the capture matrix is achieved. Furthermore, the enhanced clarification of the cell culture liquid results in reduced precipitations during affinity chromatography and thus increases the lifetimes of the expensive affinity resin. This is an important improvement for immunoglobulin purification in large scale production. The present invention further relates to sequential polishing chromatographies and filtration steps downstream to the capture chromatography, yielding immunoglobulins of high purity.