Hydroxyapatite Chromatography for Antibody Variant Purification
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Solution Overview
Problem
Existing purification methods for monoclonal antibodies and fusion proteins are inadequate in effectively removing low molecular weight aggregates (LMWs) and basic variants, leading to potential safety issues and manufacturing inefficiencies, and current chromatography techniques like ion exchange and hydrophobic interaction chromatography can induce aggregate formation or result in significant dilution.
Innovation Solution
A scalable purification process using ceramic hydroxyapatite chromatography with phosphate elution gradients to separate LMWs and basic variants, reducing impurities such as host cell proteins, DNA, and aggregates, while maintaining high purity of the target protein.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If ion exchange chromatography is used to separate antibody variants, then charge variants can be separated, but aggregates may form due to increased protein concentration or buffer concentration/pH changes during elution
Solution Approach 1:
The patent uses hydroxyapatite chromatography with phosphate buffer gradients instead of traditional ion exchange chromatography with salt gradients. This parameter change in the elution mechanism (from ionic strength-based to phosphate concentration-based) allows separation of charge variants without causing aggregate formation, as the phosphate gradient does not induce the same protein concentration increases or pH changes that promote aggregation
2Manufacturing precision
If hydrophobic interaction chromatography is used to separate protein variants, then size and charge variants can be separated, but significant dilution occurs
Solution Approach 1:
The patent employs hydroxyapatite chromatography with phosphate buffer gradients, which changes the separation parameter from hydrophobic interaction to phosphate concentration-based separation. This allows effective separation of size and charge variants while maintaining higher product concentration, avoiding the significant dilution that occurs with traditional hydrophobic interaction chromatography
3Manufacturing precision
If traditional chromatography methods are used to remove LMWs and basic variants, then purification can be achieved, but the process is cumbersome and efficiency is reduced
Solution Approach 1:
The patent uses hydroxyapatite chromatography with phosphate buffer gradients, which changes the separation mechanism to be more efficient for removing low molecular weight aggregates and basic variants. The phosphate gradient provides superior separation resolution and capacity, reducing the number of steps required and improving overall manufacturing efficiency compared to traditional chromatography methods
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 over 90% for monoclonal antibodies, significantly reducing LMWs to below 0.4% and basic variants to below 15%, ensuring compliance with regulatory standards and enhancing manufacturing efficiency.
Implementation Method 1
A scalable purification process using ceramic hydroxyapatite chromatography with phosphate elution gradients to separate LMWs and basic variants
Implementation Method 2
Ceramic hydroxyapatite (CHT) chromatography is reported for the separation of HMW by using sodium chloride or calcium chloride
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.