rHu GCSF Purification via Aqueous Two-Phase Extraction and Chromatography
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for purifying recombinant human granulocyte colony stimulating factor (rHu GCSF) from inclusion bodies in microbial cells face challenges in achieving high recovery and purity, particularly with aqueous two-phase systems, which often result in low recovery rates and high impurity levels, and conventional ion exchange chromatography is costly due to intolerance to high ionic strength solutions.
Innovation Solution
A method involving solubilization, refolding, ultra-filtration, aqueous two-phase extraction, and multimodal chromatography to purify rHu GCSF, utilizing a combination of chaotropic agents, reducing agents, and specific buffer conditions to achieve high recovery and purity, including the use of polymers and salts to form two-phase systems and subsequent chromatography steps to remove impurities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If aqueous two-phase system is used for purification of rHu GCSF, then process cost is reduced and throughput is increased, but recovery rate decreases and impurity levels increase
Solution Approach 1:
The purification process is divided into multiple sequential stages: aqueous two-phase extraction for bulk purification followed by ion exchange chromatography for polishing. This segmentation allows each stage to optimize for its specific function - ATPS for high throughput and cost-effectiveness, while chromatography for high recovery and purity
Solution Approach 2:
An intermediary buffer exchange step is introduced between ATPS and chromatography to adjust ionic strength. This mediator enables the transition from high-salt ATPS conditions to low-salt chromatography conditions, allowing both processes to operate at optimal parameters
2Manufacturing precision
If conventional ion exchange chromatography is used, then product purity is improved, but operational cost increases due to intolerance to high ionic strength solutions
Solution Approach 1:
The aqueous two-phase extraction is performed as a preliminary step to remove bulk impurities and concentrate the product before chromatography. This preliminary action reduces the load on the chromatography system, allowing it to operate more efficiently at lower costs
Solution Approach 2:
The ionic strength parameter is changed between process stages - high ionic strength in ATPS for cost-effective bulk purification, then reduced via buffer exchange to low ionic strength for chromatography to achieve high purity. This parameter change allows both processes to operate optimally
3Productivity
If prokaryotic expression system is used for rHu GCSF production, then manufacturing cost is reduced and production speed is increased, but product purity worsens due to inclusion body formation
Solution Approach 1:
The inclusion bodies are extracted and isolated from the cellular material, then solubilized and refolded to recover the active protein. This extraction approach separates the pure protein from the impure inclusion body matrix, enabling high purity recovery despite rapid production in prokaryotes
Solution Approach 2:
The mechanical refolding process replaces the need for eukaryotic post-translational modification systems. By using controlled chemical refolding conditions, the prokaryotic system's limitation (lack of glycosylation) is compensated, achieving pure functional protein without expensive eukaryotic expression
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 a recovery of over 99.96% of rHu GCSF with minimal host cell protein and DNA contamination, and subsequent chromatography steps ensure high product purity, addressing the limitations of existing techniques by enhancing recovery and reducing operational costs.
Implementation Method 1
solubilizing inclusion bodies using a solubilization buffer to obtain solubilized rHu GCSF
Implementation Method 2
refolding the solubilized rHu GCSF using a refolding buffer to obtain refolded rHu GCSF
Implementation Method 3
concentrating the refolded rHu GCSF by ultra-filtration to obtain concentrated rHu GCSF
Implementation Method 4
subjecting the concentrated rHu GCSF to aqueous two phase extraction to remove host cell proteins and host cell DNA and to obtain rHu GCSF partitioned into polymeric phase
Implementation Method 5
subjecting the rHU GCSF solution to chromatography purification to remove product related impurities and obtain purified rHU GCSF
Data Source
AI summary
The present invention relates to methods for purification of recombinant human granulocyte colony stimulating factor (rHu GCSF). The present invention particularly relates to methods for purification of rHu GCSF involving techniques such as aqueous two phase extraction and multimodal chromatographic purification to obtain highly purified rHu GCSF. The present invention also provides a pharmaceutical composition comprising the rHu GCSF, purified using the methods described herein.