Binding Protein Purification Using Sequential pH Washes
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Solution Overview
Problem
Existing methods for purifying binding proteins, such as monoclonal antibodies, are inadequate for removing undesirable components like high molecular weight aggregates and free light chains not associated with heavy chains, particularly in CHO cell cultures, leading to impure protein compositions.
Innovation Solution
Incorporating a basic wash step with a pH of at least 2.5 to 5 into the protein purification process using affinity chromatography, followed by an acidic wash and elution, to separate binding proteins from free light chains, and optionally using cation exchange chromatography for further purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional affinity chromatography methods are used to purify binding proteins, then the purification process is simple and fast, but the purity of the binding protein is insufficient due to inability to remove free light chains and aggregates
Solution Approach 1:
The purification process is segmented into multiple distinct steps: (1) affinity chromatography at neutral pH to capture binding proteins, (2) basic wash step at pH 9.0-11.0 to remove free light chains, (3) acidic wash step at pH 3.0-5.0 to remove aggregates, and (4) elution step. Each step targets specific impurities, progressively improving purity without overwhelming complexity
Solution Approach 2:
The invention utilizes parameter changes, specifically pH modulation, to achieve selective removal of different impurity types. The basic wash buffer (pH 9.0-11.0) selectively removes free light chains, while the acidic wash buffer (pH 3.0-5.0) selectively removes aggregates. This parameter-based separation enables high purity without requiring complex equipment
2Manufacturing precision
If multiple washing steps are added to remove free light chains and aggregates, then the purity of binding protein increases, but the time and complexity of the purification process increases
Solution Approach 1:
The purification process employs periodic action through sequential washing steps with different pH buffers. The basic wash step followed by acidic wash step creates a rhythmic, systematic approach to impurity removal. Each wash step is performed for a defined period, enabling efficient removal of different impurity types in sequence rather than requiring multiple iterative cycles
Solution Approach 2:
The basic wash step is performed preliminarily to remove free light chains before the acidic wash step removes aggregates. This preliminary removal of soluble impurities prepares the system for subsequent removal of aggregate impurities, making the overall process more efficient and reducing the time required for each subsequent step
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 achieves higher yields of unbound binding proteins with increased purity, allowing for more potent therapeutic compositions by effectively removing free light chains and high molecular weight aggregates.
Implementation Method 1
loading a composition comprising the binding protein and free light chain not associated with heavy chain onto an equilibrated affinity chromatography column of neutral pH to bind the binding proteins in the composition to the affinity chromatography column
Implementation Method 2
washing the affinity chromatography column with a basic wash buffer having a pH of at least 2.5 to 5 above neutral pH to wash free light chain not associated with heavy chain from the composition
Implementation Method 3
eluting binding proteins bound to the affinity chromatography column with an elution buffer
Data Source
AI summary
The present disclosure relates to a method of purifying a binding protein from undesirable components. Such binding proteins may be useful for treating a disorder such as cancer.