HIC-HPLC Antibody Quantitation Separation
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Solution Overview
Problem
Developing a method to accurately quantify individual monoclonal antibodies in co-formulated drug substances is challenging due to their similar molecular weights, protein structures, and charge properties, which hinders their separation and concentration measurement.
Innovation Solution
The method employs hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC) to separate and quantify antibodies based on differences in surface hydrophobicity, using a buffer at pH 5.0 to 7.0, and generates chromatographs with distinct peak elution times to ensure accurate quantitation of each antibody, even when they have similar molecular weights and charge properties.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional chromatography methods are used to separate antibodies, then the method is simple, but the separation precision is insufficient due to similar molecular weights, protein structures, and charge properties
Solution Approach 1:
The patent changes the separation parameter from charge-based or size-based chromatography to hydrophobicity-based HIC-HPLC. By utilizing differences in surface hydrophobicity (quantified by Kyte & Doolittle scale) rather than molecular weight or charge, the method achieves separation of antibodies with similar physical properties. This parameter transformation enables precise quantitation while maintaining operational simplicity through a straightforward chromatographic process.
2Measurement precision
If antibodies with similar molecular weights and charge properties are separated, then the quantitation accuracy improves, but the separation difficulty increases
Solution Approach 1:
The patent replaces conventional mechanical separation mechanisms (based on size exclusion or charge interaction) with a hydrophobic interaction mechanism. By substituting the separation principle to rely on hydrophobicity differences rather than physical size or charge, the method achieves accurate quantitation of similar antibodies without increasing operational difficulty. The HIC-HPLC system provides a robust mechanism that naturally exploits subtle hydrophobicity variations.
3Measurement precision
If hydrophobicity-based HIC-HPLC is used to separate antibodies, then the quantitation precision improves, but the equipment requirement becomes more specific
Solution Approach 1:
The patent demonstrates that HIC-HPLC can universally apply to separate and quantify various antibody types (monoclonal, polyclonal, different isotypes) based on their hydrophobicity differences. The method's universality allows it to handle diverse antibody formulations without requiring method re-development, achieving precise quantitation across different antibody types while using a single robust platform.
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 approach allows for precise, accurate, and reproducible quantitation of antibodies in co-formulated compositions, ensuring high purity and effective separation, even when antibodies have similar molecular weights and charge properties, making it suitable for quality control environments without requiring expensive equipment or cumbersome sample preparation.
Implementation Method 1
separating each of the plurality of antibodies in the mixture using hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC)
Data Source
AI summary
The present disclosure relates to, inter alia, a method of quantitating an amount of an antibody molecule from a mixture comprising two or more antibody molecules, comprising separating each of the two or more antibody molecules from the mixture by hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC) and quantitating an amount of each antibody molecule, wherein the molecular weight of each antibody molecule is within 15 kDa of any other antibody molecule in the mixture and either each antibody molecule is different from another antibody molecule in the mixture by more than about 0.25 unit on the Kyte & Doolittle hydropathy scale or each of the antibody molecules when nm alone on HIC-HPLC elutes at distinct run time with little overlap from the other antibody molecules in the mixture, or both.