Protein Purification With Hydroxyapatite for Aggregate and Variant Removal
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Solution Overview
Problem
Existing purification methods for monoclonal antibodies and fusion proteins are inadequate in removing low molecular weight aggregates and basic variants, leading to potential safety issues and manufacturing inefficiencies, and current chromatographic techniques can induce aggregate formation or result in significant product dilution.
Innovation Solution
A scalable purification process using ceramic hydroxyapatite chromatography with phosphate elution gradients to separate and reduce low molecular weight aggregates and basic variants, without the use of NaCl or CaCl2, achieving high purity levels of monoclonal antibodies and fusion proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If ion exchange chromatography is used to separate charge variants, then charge separation is achieved, but aggregate formation is induced due to increased protein concentration or buffer concentration/pH changes during elution
Solution Approach 1:
The patent uses phosphate buffer gradients instead of salt-based elution, changing the chemical parameter of the elution medium to avoid aggregate formation while maintaining charge variant separation capability
Solution Approach 2:
The patent introduces phosphate ions as an intermediary substance that enables selective binding and elution of charge variants without causing the harmful effects of traditional salt-based ion exchange chromatography
2Manufacturing precision
If size exclusion chromatography is used to separate molecular weight variants, then size-based separation is achieved, but significant product dilution occurs
Solution Approach 1:
The patent changes the separation parameter from size-based to charge-based using phosphate buffer gradients, which allows for more efficient separation without the dilution problem inherent in size exclusion chromatography
3Manufacturing precision
If affinity chromatography is used to purify antibodies, then high specificity is achieved, but ligand leakage contaminates the eluted product
Solution Approach 1:
The patent uses phosphate buffer as an intermediary that selectively interacts with the antibody charge properties, enabling purification without the ligand leakage issue of affinity chromatography
4Manufacturing precision
If conventional chromatography is used to remove low molecular weight aggregates, then some purification is achieved, but basic variants remain and complement activation may occur
Solution Approach 1:
The patent changes the chromatographic parameter to phosphate buffer gradients, which provides superior separation of both low molecular weight aggregates and basic variants, completely removing harmful factors that could activate complement
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
The process achieves purity levels of 90% or higher for monoclonal antibodies and fusion proteins, significantly reducing low molecular weight aggregates and basic variants, thereby enhancing product safety and manufacturing efficiency.
Implementation Method 1
A scalable purification process using ceramic hydroxyapatite chromatography with phosphate elution gradients to separate and reduce low molecular weight aggregates and basic variants
Implementation Method 2
the present invention uses anion exchange which significantly reduce high molecular weight impurity and acidic variants
Data Source
AI summary
The invention provides a process of purification of antibody or fusion protein from protein mixture comprising product and process related impurities. The process provides the use of hydroxyapatite chromatography for the separation of low molecular weight impurities and basic variants. In addition, invention further provides a scalable purification process to remove product and process related impurities.