Ranibizumab Purification via Multimodal Chromatography
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Solution Overview
Problem
Current downstream purification processes for recombinant humanized Ranibizumab from E. coli cells are cumbersome and inefficient, leading to high costs and low throughput due to multiple purification steps, particularly when dealing with inclusion bodies that form insoluble protein aggregates.
Innovation Solution
A process integrating multimodal chromatographic purification steps, including solubilization, refolding, ultra-filtration, and sequential multimodal chromatography to remove impurities, with specific buffer conditions and pH ranges to achieve high purity and activity standards, applicable to both in-vitro refolded and soluble expressed antibody fragments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If multiple purification steps (ultrafiltration, diafiltration, anion exchange chromatography, cation exchange chromatography) are used to purify Ranibizumab from inclusion bodies, then purity is improved, but process complexity and time increase significantly
Solution Approach 1:
The patent combines multiple purification techniques (ultrafiltration, diafiltration, anion exchange chromatography, cation exchange chromatography) into a single integrated downstream processing platform. This merging of operations reduces the number of separate unit operations while maintaining high purity levels, directly addressing the contradiction between purity and process complexity
Solution Approach 2:
The downstream processing platform is designed to handle multiple purification functions simultaneously - concentration, buffer exchange, and chromatographic purification - making it a universal system that can achieve multiple objectives in a unified process, thereby reducing overall process complexity while maintaining manufacturing precision
2Manufacturing precision
If multiple purification steps are implemented to remove impurities from Ranibizumab, then purity is improved, but productivity decreases due to low throughput
Solution Approach 1:
The integrated downstream processing platform enables continuous operation where ultrafiltration, diafiltration, and chromatographic steps are seamlessly connected without intermediate interruptions. This continuity maintains high throughput while achieving the necessary purity levels, resolving the contradiction between purity and productivity
Solution Approach 2:
By merging multiple purification operations into a single integrated platform, the system eliminates the sequential bottlenecks that would otherwise reduce throughput. The combined operations process material in a streamlined fashion, maintaining high productivity while ensuring impurity removal
3Manufacturing precision
If conventional purification processes are used for Ranibizumab, then purity can be achieved, but manufacturing cost increases due to extensive chromatographic operations
Solution Approach 1:
The patent merges multiple chromatographic operations (anion exchange, cation exchange) with ultrafiltration and diafiltration into a single integrated platform, reducing the total number of expensive chromatographic steps required while maintaining high purity, thereby lowering manufacturing costs
Solution Approach 2:
The integrated platform utilizes the properties of the protein and impurities themselves to facilitate purification - using size differences for ultrafiltration, charge differences for chromatography - reducing the need for additional expensive reagents and materials, thus lowering manufacturing costs while achieving high purity
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 results in a nearly two-fold improvement in productivity and achieves high purity with less than 5% unfolded or misfolded forms and less than 2% aggregated forms of Ranibizumab, meeting innovator product standards and being applicable to both in-vitro refolded and soluble expressed antibody fragments.
Implementation Method 1
solubilizing inclusion bodies using solubilization buffer to obtain solubilized rHu Ranibizumab
Implementation Method 2
refolding solubilized rHu Ranibizumab using refolding buffer to obtain refolded rHu Ranibizumab
Implementation Method 3
concentrating refolded rHu Ranibizumab by ultra-filtration to obtain concentrated rHu Ranibizumab
Implementation Method 4
subjecting concentrated rHu Ranibizumab to sequential multimodal chromatography purification steps to remove product related impurities, host cell proteins and host cell nucleic acids
Data Source
AI summary
The present invention relates to a purification process of recombinant antibody fragments from inclusion bodies expressed in microbial cells. More particularly, the present invention relates to a process for purification of recombinant humanized (rHu) antibody fragment, Ranibizumab from inclusion bodies expressed in microbial cells.