Antibody Purification via Mixed Mode and Hydroxyapatite Chromatography
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for antibody purification, such as affinity chromatography using Protein A, are costly and inefficient, and there is a need for a more effective and cost-effective method to achieve high purification of antibodies without significant aggregates or cellular contaminants.
Innovation Solution
A method involving mixed mode chromatography using a matrix with hydrophobic and acidic moieties, followed by hydroxyapatite or fluorapatite chromatography, which binds and elutes antibodies effectively, reducing the need for sample manipulation and allowing for high purification at a lower cost.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If Protein A affinity chromatography is used for antibody purification, then high purification is achieved, but the cost increases significantly
Solution Approach 1:
The purification process is divided into two sequential chromatography steps: first using mixed mode chromatography to capture and concentrate antibodies, then using hydroxyapatite or fluorapatite chromatography for final polishing. This segmentation allows each step to be optimized for its specific function, achieving high purification without relying solely on expensive Protein A affinity chromatography throughout the entire process
Solution Approach 2:
The patent employs alternative chromatography media (mixed mode and hydroxyapatite/fluorapatite) that are less expensive than Protein A affinity media. These media provide sufficient purification capability at lower cost, effectively replacing the expensive Protein A step while maintaining product quality
2Manufacturing precision
If multiple purification steps are used to remove aggregates and cellular contaminants, then purification quality improves, but the process complexity increases
Solution Approach 1:
The patent combines capture, concentration, and initial purification functions into a single mixed mode chromatography step, then uses hydroxyapatite or fluorapatite chromatography for final polishing in a second step. This merging of functions reduces the total number of separate purification steps compared to traditional multi-step protocols while maintaining high purification quality
Solution Approach 2:
The mixed mode chromatography matrix is designed to perform multiple functions simultaneously: it captures antibodies through affinity interactions, concentrates them through binding, and begins purification by removing some contaminants. This multi-functionality reduces the need for separate dedicated steps for each function, simplifying the overall process
3Manufacturing precision
If extensive sample manipulation is performed during purification, then purification quality improves, but the time and operational complexity increase
Solution Approach 1:
The mixed mode chromatography step is designed to perform preliminary capture, concentration, and initial purification in a single operation before the final hydroxyapatite or fluorapatite polishing step. This preliminary action prepares the sample in advance, reducing the need for additional intermediate manipulation steps and minimizing processing time while maintaining high purification quality
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 highly purified antibodies with reduced costs and eliminates the need for additional purification steps, suitable for both large-scale commercial and laboratory-scale preparations, while effectively removing contaminants and aggregates.
Implementation Method 1
Mixed mode chromatography involves the use of solid phase chromatographic supports that employ multiple chemical mechanisms to adsorb proteins or other solutes. Examples include chromatographic supports that exploit combinations of two or more of the following mechanisms: anion exchange, cation exchange, hydrophobic interaction, hydrophilic interaction, hydrogen bonding
Implementation Method 2
chromatographic supports that exploit combinations of two or more of the following mechanisms: anion exchange, cation exchange
Implementation Method 3
chromatographic supports that exploit combinations of two or more of the following mechanisms: anion exchange, cation exchange, hydrophobic interaction, hydrophilic interaction, hydrogen bonding
Implementation Method 4
contacting the solution comprising eluted antibodies of step c to a solid support matrix, the solid support matrix comprising hydroxyapatite (HT) or fluorapatite (FT), wherein at least a portion of the antibodies bind to the solid support matrix
Data Source
Figure 1
Figure 2
Figure 3
AI summary
Methods, kits and apparatuses for chromatography purification of antibodies are provided. In some embodiments, antibodies are purified by mixed mode chromatography that does not comprise hydroxyapatite (HT) or fluorapatite (FT). The mixed mode chromatography step is then followed by a HT/FT chromatography step.