Multi-Domain Protein Charge Balancing for Native Colloidal Stability
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Solution Overview
Problem
Existing methods for predicting protein aggregation propensity in multi-domain proteins are biased and do not accurately reflect native state stability due to reliance on stress conditions that unfold the molecule, and there is a need for methods to minimize aggregation and extend long-term stability.
Innovation Solution
A method involving calculating net charge and introducing modifications to minimize charge sign differences between domains, optionally adjusting hydrophobicity, to produce multi-domain proteins with improved colloidal stability, using a linear sequence and homology model to predict aggregation propensity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If stress conditions (heat, etc.) are applied to evaluate protein aggregation, then aggregation propensity can be detected, but the protein is partially unfolded exposing hydrophobic core and predictions become biased towards hydrophobic model
Solution Approach 1:
The patent applies preliminary anti-action by using PEG-induced precipitation to prevent protein unfolding before aggregation evaluation. PEG creates crowding effects that promote aggregation of natively folded proteins without requiring heat stress, thereby preventing the exposure of hydrophobic cores and maintaining native state structure during the assessment process.
Solution Approach 2:
The patent introduces PEG as an intermediary substance that mediates the aggregation process. Instead of applying direct heat stress that unfolds proteins, PEG acts as a crowding agent that induces aggregation of natively folded proteins, allowing evaluation of aggregation propensity while preserving native state structure.
2Measurement precision
If ammonium sulfate precipitation is used to study colloidal interactions, then precipitation can be induced, but high salt concentrations perturb native electrostatic interactions between protein molecules
Solution Approach 1:
The patent uses PEG as an intermediary crowding agent instead of ammonium sulfate. PEG induces precipitation through excluded volume effects without introducing high salt concentrations, thereby maintaining native electrostatic interactions between protein molecules while still enabling the study of colloidal interactions.
3Measurement precision
If existing aggregation prediction methods are used, then aggregation propensity can be estimated, but they are biased towards hydrophobic model and do not accurately predict native state colloidal stability
Solution Approach 1:
The patent changes the fundamental parameter used for aggregation prediction from hydrophobicity-based metrics to charge-based metrics. By calculating the difference in net charge between domains and using this to predict aggregation propensity through PEG-induced precipitation, the method achieves accurate prediction of native state colloidal stability without the biases of hydrophobic models.
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 method effectively predicts and reduces colloidal aggregation propensity, enhancing the stability of multi-domain proteins without external stress, validated at biologically relevant pH values.
Implementation Method 1
The methods of the present invention allow to predict the colloidal aggregation propensity of multi-domain molecules based on the frequency of charge interactions between the domains of native folded molecules
Implementation Method 2
PEG induced precipitation assays are more often used to study colloidal interactions as ammonium sulphate precipitation requires the addition of high salt concentrations which can perturb native electrostatic interactions between protein molecules
Data Source
AI summary
The present invention provides a method for generation of multi-domain proteins, more particular antibodies, with an improved native state colloidal stability. The present disclosure provides a method of producing an IgG1 or IgG4 antibody with an improved colloidal stability comprising, in embodiments: calculating for each domain of said IgG1 or IgG4 antibody the total net charge at a given pH; introducing one or more modifications to the amino acid residues of the constant region of said IgG1 or IgG4 antibody to minimize the charge sign difference between the domains, wherein said one or more modification is, or each are, a substitution of a charged amino acid by a polar (non-charged) amino acid; and producing the modified multi-domain protein with improved colloidal stability at the given pH.