Correctly folded etanercept in high purity and excellent yield

a technology of etanercept and folds, applied in the field of chromatographic separation methods for purifying recombinantly expressed proteins, can solve the problems of large amount of incorrectly or misfolded products produced by cho cells, similar loss of therapeutic effect, and detrimental to patients, so as to maximize the stability and solubility of etanercept, minimize discomfort to patients, and maximize the effect of active ingredient stability

Inactive Publication Date: 2014-03-13
COHERUS BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention relates to a method of producing a high purity etanercept preparation through a mixed mode chromatography process. This method allows for the reduction or elimination of a specific peak in the HIC chromatogram, which is believed to contain incorrectly folded etanercept. The invention also provides pharmaceutical formulations of etanercept using high purity etanercept obtained through this method and commonly used buffers and excipients. The resulting formulations have improved stability and solubility, and the pH of the composition can be adjusted for optimal stability and tolerability for patients.

Problems solved by technology

Unfortunately, the product that is produced by the CHO cells contains a large amount of incorrectly or misfolded and / or aggregated etanercept.
For pharmaceutical use, it is desirable to provide etanercept that is relatively free of incorrectly folded and aggregated protein because the incorrectly folded / aggregated protein will not have the same therapeutic effect as the correctly folded protein, and may actually be detrimental to the patient.
Aggregation, generally understood to involve non-covalent association of two or more etanercept homodimers to form very high molecular weight species, results in a similar loss of therapeutic effect, and occurs when proteins, including misfolded proteins, accumulate and clump together.
As stated above, such misfolded proteins and protein aggregates are not only therapeutically ineffective, but may also be detrimental to the patient.
There are many therapeutic proteins for which misfolding may be a problem.
The use of low pH and organic solvent in reverse-phase chromatography, however, can denature the proteins and may cause aggregation of the purified protein during the chromatography.
Prior teachings such as those referenced above have not proven useful for application to the separation of correctly folded etanercept from incorrectly folded etanercept in that they fail to provide qualitatively and / or quantitatively adequate samples.

Method used

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  • Correctly folded etanercept in high purity and excellent yield
  • Correctly folded etanercept in high purity and excellent yield
  • Correctly folded etanercept in high purity and excellent yield

Examples

Experimental program
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Effect test

example 1

Capto™ MMC Mixed Mode Purification Using NaCl Elution

[0139]In this example, a Protein A eluate obtained as described above is subjected to Capto™ MMC chromatography. The Capto™ MMC column is equilibrated with a 5 mM citrate, pH 4.5 solution. An appropriate dilution of Protein A eluate (e.g., 3 to 6 fold dilution depending on the elution buffer of Protein A step) with 5 mM citrate results in complete binding to the column. As was shown in SDS-PAGE analyses, the loading sample (pH 4.2, Protein A eluate) is highly heterogeneous and no protein flows through the Capto™ MMC column. The bound proteins is then eluted with a 0.15 M NaCl in 10 mM phosphate, pH 7.5 solution which leads to a simultaneous change in pH (from 4.5 to 7.5) and NaCl concentration (from 0 to 0.15 M). A sharp elution peak containing correctly folded etanercept is observed from analysis of the eluate. However, the recovery was about 40-50%. Following the elution step as just described, the resin containing the remaining...

example 2

Capto™ MMC Mixed Mode Purification Using NaCl Resin Equilibrated to pH 7.5

[0140]In the foregoing example, the protein solution comprising correctly folded and incorrectly folded etanercept was initially contacted with the CAPTO MMC resin at pH 4.5. In this example, a similar elution to that observed in Example 1 is observed when the Capto™ MMC column is first equilibrated with a 10 mM phosphate, pH 7.5 solution and then eluted with NaCl followed by arginine. Specifically, no etanercept is eluted during this pH equilibration. This result is unexpected because etanercept is negatively charged at pH 7.5; the pI of etanercept ranges from 4.9 to 5.4 due to heavy glycosylation. Thus, at or below pH 4.5, etanercept is positively charged and hence should bind to the negatively charged Capto™ MMC ligands, although hydrophobic interaction may contribute to the binding. At pH 7.5, the negatively charged etanercept should dissociate from the negatively charged Capto™ MMC, but it is found that t...

example 3

Capto™ MMC Mixed Mode Purification Using NaCl Gradient for Elution

[0141]In this example, successful elution is accomplished using an NaCl concentration gradient. Specifically, after washing the column with the 10 mM phosphate, pH 7.5 solution, the bound proteins were eluted with linear salt gradient from 0 to 0.5 M. The loading sample (pH 4.2 Protein A eluate) is extremely heterogeneous (containing both correctly folded and incorrectly folded etanercept) as in the previous case, and the bound proteins are eluted with increasing ionic strength of the NaCl gradient. After 180 min, the gradient is terminated and salt concentration is then brought to 0.5 M, which caused a slightly enhanced protein elution. As determined by subsequent analyses, low salt fractions contain more of the correctly folded etanercept and higher salt fractions were enriched with low mobility (misfolded and aggregated) species. Finally, the remaining proteins is eluted with a 1 M arginine, 10 mM phosphate, pH 7.5...

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Abstract

A mixed mode chromatography method for separating correctly folded from incorrectly folded conformations of a given protein is provided. The method is highly effective in separating correctly folded etanercept from incorrectly folded etanercept and aggregates in commercially attractive yields capable of affording etanercept preparations having very high purity in terms of correctly folded etanercept versus incorrectly folded etanercept. The invention is further directed to protein preparations and formulations comprising correctly folded proteins obtained using the present methods, and methods of treatment using the high purity preparations obtained from the mixed mode method.

Description

FIELD OF THE INVENTION[0001]The present invention relates generally to chromatographic separation methods for purifying recombinantly expressed proteins, and products obtained from such methods. More particularly, it relates to use of mixed mode chromatography to purify a recombinant protein expression product, including, for example, fusion proteins which may include undesired amounts of incorrectly folded and / or aggregated protein along with properly folded protein. The mixed-mode chromatography method of the invention is especially useful for separating correctly folded etanercept from incorrectly folded etanercept (as defined herein). The invention is also directed to etanercept preparations and pharmaceutical formulations wherein the etanercept supplied therein has been produced in high yield and high purity using the disclosed method. The invention further concerns treatment methods for TNF conditions employing highly purified etanercept characterized by remarkably low levels ...

Claims

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Application Information

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IPC IPC(8): C07K16/24C07K14/705
CPCC07K14/70578C07K16/241C07K1/165C07K2319/30A61P17/06A61P19/02A61P29/00A61P37/02A61K38/16A61K39/395A61K2039/505
Inventor ARAKAWA, TSUTOMUFARRAR, DOUGLAS
Owner COHERUS BIOSCI
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