Hydroxyapatite Chromatography for Conjugate Purification
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Solution Overview
Problem
Current methods for purifying saccharide antigen-carrier protein conjugates are inefficient, particularly for conjugates with saccharide antigens of low molecular weight, as they require large volumes of gel filtration matrix or result in low yield and stress the conjugate, and existing ultrafiltration methods are not effective for all types of conjugates.
Innovation Solution
The method involves contacting a mixture of saccharide antigen-carrier protein conjugates with hydroxyapatite, where unconjugated carrier proteins bind, allowing for the purification of saccharide antigen-carrier protein conjugates while optionally reusing or discarding the unbound carrier protein.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If gel filtration is used to purify conjugates, then purification can be achieved, but large volume of gel filtration matrix is required and it is difficult to apply at manufacturing scale
Solution Approach 1:
The invention changes the binding parameters of carrier proteins by using hydroxyapatite chromatography conditions where unconjugated carrier proteins bind to the hydroxyapatite matrix while conjugated forms do not bind and pass through in the flow-through fraction. This parameter-based separation eliminates the need for large volumes of gel filtration matrix while achieving effective purification at manufacturing scale
Solution Approach 2:
The invention extracts and removes unconjugated carrier proteins from the conjugate mixture by binding them to hydroxyapatite, separating them from the conjugated products that remain in the flow-through. This extraction approach achieves purification without requiring large volumes of filtration matrix
2Manufacturing precision
If ultrafiltration with 100 KDa membrane is used, then purification can be achieved for high molecular weight saccharide antigens, but it is not effective for GBS serotype III conjugates or other conjugates where the saccharide antigen is of low molecular weight
Solution Approach 1:
The hydroxyapatite chromatography method provides universal purification capability for all conjugate types regardless of saccharide antigen molecular weight. The method works for both high and low molecular weight conjugates including GBS serotype III, making it a versatile multi-functional purification approach that replaces molecular weight-dependent ultrafiltration methods
3Manufacturing precision
If conventional purification methods are used, then free carrier protein can be removed, but the methods stress the conjugate and result in low yield
Solution Approach 1:
Instead of trying to retain the conjugate and remove impurities, the invention inverts the approach by allowing unconjugated carrier proteins to bind to hydroxyapatite while the conjugated products pass through unbound in the flow-through. This inverted binding approach minimizes stress on the conjugate and maximizes yield
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 effectively purifies saccharide antigen-carrier protein conjugates by selectively binding unconjugated carrier proteins to hydroxyapatite, achieving high yields and stability of the conjugates, especially for conjugates with low molecular weight saccharide antigens, and allows for the reuse of unbound carrier proteins.
Implementation Method 1
the contaminant/unconjugated proteins bind to hydroxyapatite while saccharide antigen-carrier protein conjugates do not substantially bind
Data Source
AI summary
This application relates to methods for the purification of saccharide antigen-carrier protein conjugates. In particular, the invention provides a method for purifying saccharide antigen-carrier protein conjugates from free carrier protein, such as CRM1 97, using hydroxyapatite. The invention further relates to methods of preparing vaccines, using this method.