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A method for improving aggregate removal by protein a chromatography

A chromatography and protein technology, applied in the field of protein A chromatography to remove antibody aggregates, to reduce the burden, improve the removal ability, and improve the overall robustness

Pending Publication Date: 2021-07-23
SHANGHAI WUXI BIOLOGIC TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This reliance on single-step designs is especially problematic for projects with above-average aggregate levels

Method used

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  • A method for improving aggregate removal by protein a chromatography
  • A method for improving aggregate removal by protein a chromatography
  • A method for improving aggregate removal by protein a chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1: Effect of PEG on protein A elution curve

[0074] In this study, we first investigated the effect of PEG on the eluting curve of the protein A by adding different amounts of PEG (ie 1.5%, 3%, 5% and 10%) to the cleaning and elution buffer. With the increase in PEG concentration, the retention rate of easy-to-gathered antibodies is slightly improved, and eluting peaks become sharp ( figure 1 ). However, different from other types of columns (eg, ion exchange, hydrophobic interaction, and mixing mode), PEG (up to 10%) has no effect on the monomer-aggregate separation of the protein A column. This observation explained the previous lack of reports on the application of PEG to aggregates in protein A.

Embodiment 2

[0075] Example 2: Effect of Calcium chloride on the eluting curve of protein A

[0076] The present invention designs experiments to explore calcium chloride as a liquid phase additive to the separation of monomer - aggregate. For the case studied, different amounts of calcium chloride (ie 250 mm, 500 mM, 750 mM, and 1M) were added to the protein A cleaning and elution buffer.

[0077] Adding Calcium chloride to the flow phase showed awareness but not significant effect on the separation and retention time ( figure 2 ). At low concentration (ie 250 mM), calcium chloride has little effect on separation, and eluting peaks are similar to the elution peaks that do not contain the salt (in both cases, eluting peaks are relatively sharp) ). However, calcium chloride in this concentration increases the retention time of the target protein. At an increased concentration (i.e., 500 mM and 750 mM), calcium chloride showed a small amount of impact on the separation. Under these two condition...

Embodiment 3

[0079] Example 3: Synergistic effect of PEG and calcium chloride on protein A separation

[0080] Although calcium chloride increases the monomer-aggregate separation at 500 mm and 750 mm, the separation of two substances under these conditions is still not complete enough. Therefore, the inventors will then attempt to combine PEG / chloride. Since PEG itself has little effect on elution curve at different concentrations, the inventors in this study have arbitrarily selected 5% PEG and different amounts of calcium chloride. In the calcium chloride concentration (i.e., 150 mm and 250 mm), this combination did not show significant effects, and the elution curve was almost consistent with only 5% PEG. Figure 4 A). However, the combination of 500 mM Calcium chloride and 5% PEG exhibits a stringent synergistic effect, resulting in a significant increase in the separation of monomers and aggregates ( Figure 4 B). The monomers in the eluent were raised from 80% (cleaning 2 and the elutio...

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Abstract

Protein A chromatography is generally less effective in removing antibody aggregates under typical conditions. Provided is a combination and a method that can significantly improve Protein A's aggregate removal capability. The combination comprises polyethylene glycol (PEG) and a salt (chaotropic or kosmotropic) as wash and elution buffer additives. The synergistic effect of salt and PEG results in almost complete separation of monomer from aggregates. For the case used for demonstration, in comparison with the control run the optimized procedure reduces aggregates in elution pool from 20% to 3-4%. This new method, by facilitating aggregate removal at the capture step, improves the overall robustness of downstream process.

Description

Technical field [0001] The present invention generally relates to a combination and method of removing antibody aggregates in protein a chromatography. Background technique [0002] In general, protein A chromatography is poor in the removal of aggregate effects under classic conditions. Although the polymeric group is known to be more combined with the monomer (D. Y, Y.Song, Ryhuang et al, "Antibody aggregate and the adsorption of the adsorption thereof on the protein A resin" (Molecular Perspective of Antibody Aggregates and Their Adsorptionon Protein A Resin, J.chromatogr.a, 2016, 1457, 66-75), but they often elute together with the latter, and separately adjusting the eluting pH is usually not enough to achieve a good separation effect. Therefore, there are many In case, the removal of the aggregate relies on the single refining chromatography after protein A. However, the removal of the aggregate relying on a single step is unfavorable because such design reduces the robustn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/36C07K1/16C07K1/22C07K1/18C07K16/00A61K39/395
CPCA61K39/395C07K1/22C07K16/00B01D15/3809B01D15/426
Inventor 李翊峰王影张远周伟昌
Owner SHANGHAI WUXI BIOLOGIC TECH CO LTD
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