IL-15/IL-15Ralpha Conjugate Purification via Inverted Chromatography
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Solution Overview
Problem
Current methods for producing IL-15/IL-15Ralpha conjugates result in dimeric and multimeric complexes, leading to immunogenicity and allergenicity concerns, making it challenging to obtain a purified composition suitable for pharmaceutical use due to their short half-life and low yields in mammalian cell lines.
Innovation Solution
A purification method involving anion-exchange chromatography followed by hydrophobic interaction chromatography, with specific loading procedures, is employed to obtain monomeric conjugates, enhancing refolding and reducing co-precipitation on chromatographic resins, thereby achieving high concentrations of active fusion proteins in a monomeric form.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If hydrophobic interaction chromatography is used first followed by anion-exchange chromatography, then purification is achieved, but the conjugate forms dimeric and multimeric complexes leading to immunogenicity
Solution Approach 1:
The patent inverts the conventional purification sequence by performing anion-exchange chromatography first, then hydrophobic interaction chromatography. This reversal prevents dimeric and multimeric complex formation that occurs with the traditional sequence, thereby eliminating immunogenicity while maintaining purification effectiveness.
Solution Approach 2:
The patent modifies the loading procedure parameter by directly mixing the first eluate with loading buffer into a mixing chamber of the HIC column, rather than conventional loading methods. This parameter change enables efficient monomeric form recovery without co-precipitation on the chromatographic resin, resolving the contradiction between purification and immunogenicity.
2Manufacturing precision
If conventional purification methods are used, then some purification is achieved, but dimeric and oligomeric aggregates form at high percentages making the composition unusable as medicament
Solution Approach 1:
By inverting the chromatography sequence (anion-exchange first, then HIC), the patent achieves both high purification levels and maintains medicament suitability. The inverted sequence specifically prevents aggregate formation while ensuring monomeric conjugate recovery, making the composition reliable for pharmaceutical use.
Solution Approach 2:
The patent introduces a mixing chamber as an intermediary component between the anion-exchange and HIC steps. This intermediary allows direct mixing of the first eluate with loading buffer, facilitating smooth transition and preventing aggregate formation during the purification process, thereby ensuring medicament suitability.
3Quantity of substance
If IL-15 is produced in mammalian cell lines, then the cytokine is obtained, but production yields are low due to short half-life
Solution Approach 1:
The patent merges IL-15 with IL-15Ralpha to create a conjugate that combines the cytokine with its receptor. This merging extends the half-life of IL-15 by preventing its rapid clearance, thereby increasing both the duration of action and production yield suitable for pharmaceutical applications.
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 composition with at least 80% monomeric conjugates, reducing immunogenicity and increasing the half-life, allowing for the use of IL-15/IL-15Ralpha conjugates as a medicament for treating cancer and infections.
Implementation Method 1
a purification process using anion-exchange chromatography followed by a hydrophobic interaction chromatography enables to obtain the conjugate in a monomeric form
Implementation Method 2
a purification process using anion-exchange chromatography followed by a hydrophobic interaction chromatography enables to obtain the conjugate in a monomeric form
Data Source
AI summary
The present invention relates to a method for preparing a composition comprising monomeric conjugates from a sample, said conjugate comprising (a) a polypeptide comprising the amino acid sequence of interleukin 15 or derivatives thereof, and (b) a polypeptide comprising the amino acid sequence of the sushi domain of IL-15Rα or derivatives thereof; wherein said method comprises the use of anion-exchange chromatography followed by a hydrophobic interaction chromatography; and to a pharmaceutical composition which can be obtained by such a method.