Protein Complex Characterization Using A4F-MALLS Separation
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Solution Overview
Problem
Existing methods like dynamic light scattering (DLS) and size exclusion chromatography (SEC) are inadequate for fully characterizing the size, shape, and heterogeneity of large protein complexes, particularly those formed by monoclonal antibodies, due to biases and limitations in resolving polydisperse samples and sample adsorption.
Innovation Solution
The use of asymmetrical flow field flow fractionation (A4F) combined with multi-angle laser light scattering (MALLS) to fractionate and characterize protein complexes, determining stoichiometry, size distribution, and conformation of protein:ligand complexes, including monoclonal antibodies and fusion proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If dynamic light scattering (DLS) is used to determine protein size, then average radius and polydispersity can be determined, but results are biased towards larger particles and particle populations must differ by a factor of at least three to be resolved
Solution Approach 1:
The patent combines asymmetrical flow field-flow fractionation (A4F) with multi-angle laser light scattering (MALLS) to create a hybrid system that merges the separation capability of A4F with the detection capability of MALLS, thereby overcoming the limitations of DLS alone in resolving polydisperse protein complexes
Solution Approach 2:
The patent introduces A4F as an intermediary separation technique between sample injection and light scattering detection. This intermediary step fractionates protein complexes by size before detection, enabling MALLS to accurately measure size distribution without the biases inherent in direct DLS measurement
2Manufacturing precision
If size exclusion chromatography (SEC) is used for protein separation, then separation according to hydrodynamic volume occurs, but sample adsorption to stationary phase and shear degradation at high pressures limit its effectiveness
Solution Approach 1:
The patent replaces the mechanical stationary phase-based separation of SEC with a field-based separation approach using asymmetrical flow field-flow fractionation. This substitution eliminates sample adsorption to stationary phase while maintaining separation based on hydrodynamic volume, and operates at low pressures to prevent shear degradation
Solution Approach 2:
The patent changes the separation mechanism from stationary phase interaction (SEC) to field-flow fractionation based on diffusion coefficients (A4F). This parameter change in the separation mechanism eliminates the harmful effects of adsorption and high pressure while preserving size-based separation capability
3Manufacturing precision
If flow field-flow fractionation (FFF) is used for separation, then analytes can be separated in a wide size range with reduced sample loss, but there is still a growing need to more fully characterize heterogeneity and conformation of protein complexes
Solution Approach 1:
The patent uses multi-angle light scattering to measure not only the average size but also the full size distribution and molar mass of protein complexes. This excessive measurement capability (measuring multiple parameters beyond simple size) provides comprehensive characterization of heterogeneity and conformation that FFF alone cannot provide
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
Provides detailed characterization of protein complexes, enabling selection of lead protein drug products that minimize large, heterogeneous complexes, improving pharmacokinetics and reducing immunogenicity.
Implementation Method 1
FFF sample separation uses a flow-assisted separation and fractionation method in which the analytes are separated along a ribbon like channel by differences in their diffusion coefficients
Implementation Method 2
The Brownian motion of proteins in solution causes light to be scattered, with the resultant scattered intensity fluctuations dependent on particle size
Implementation Method 3
The Brownian motion of proteins in solution causes light to be scattered, with the resultant scattered intensity fluctuations dependent on particle size
Data Source
AI summary
Methods for characterizing protein complexes formed between protein drug products and soluble ligands are provided herein. The disclosed methods can determine the size, heterogeneity, and conformation of protein complexes.