Cell culture process for making a glycoprotein

Inactive Publication Date: 2019-01-10
REGENERON PHARM INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text discusses the discovery that the concentration of certain amino acids in soy hydrolysate can affect the quality and composition of proteins produced in cell culture. By adding a specific concentration of ornithine or putrescine to the soy hydrolysate, cells can produce higher quality proteins with consistent glycosylation patterns. This method can be used to produce glycoproteins, such as trap molecules, with a desired glycan profile. The invention also includes a method for selecting a soy hydrolysate based on the amount of ornithine or putrescine present.

Problems solved by technology

However, protein hydrolysates may contain compounds that negatively impact cell growth or recombinant protein production.
However, the quality and performance of biological therapeutics are highly dependent upon the manufacturing process.
Such variation increases heterogeneity of protein isoforms in the resulting protein production, which can alter stability, efficacy or immunogenicity of the biological therapeutic and ultimately lead to the rejection of the lot of proteins.

Method used

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  • Cell culture process for making a glycoprotein
  • Cell culture process for making a glycoprotein
  • Cell culture process for making a glycoprotein

Examples

Experimental program
Comparison scheme
Effect test

example 1

Soy Hydrolysate to Determine Amino Acid Concentration

[0115]A soy hydrolysate sample was weighed and a 20 gram portion thereof was dissolved in 1 L water to a starting concentration of 20 g / L. The resulting soy hydrolysate solution was then further diluted in water to a desired concentration for use in cell culture and the molecular composition of the resulting soy hydrolysate solution was determined using by chromatography.

[0116]The concentration of amino acids in the soy hydrolysate sample was measured by chromatography on an ion exchange column with post column ninhydrin detection. See, e.g., Moore and Stein. J. Biol. Chem. (1954) Vol. 211 pp. 907-913. Soy hydrolysate samples were diluted to permit sensitive separation and resolution of individual peaks (amino acids) as eluted from the HPLC column and compared to a standard. Each peak area of the chromatogram, as shown in FIGS. 1A and 1B, was compared to a standard to determine concentration of each eluate.

[0117]To determine wheth...

example 2

n and Glycosylation Profile of a Protein of Interest

[0118]CHO cells expressing a trap protein (receptor-Fc fusion protein, VEGF-trap) were cultured in proprietary medium including soy hydrolysate containing varying amounts of ornithine, putrescine and citrulline, or a combination thereof, in order to determine which amino acid components affect the quality of proteins produced. Table 2, shows that the levels of ornithine in the hydrolysate correlate negatively with the quality of protein production lots, as indicated by the increased area under the curve for a key N-glycan for protein lots produced as a result of culturing CHO cells in media supplemented with soy hydrolysate containing ornithine at a concentration less than 5.0 mg / L independent of citrulline concentration.

[0119]As shown in Table 2 and depicted in FIG. 3, VEGF-trap protein product lots produced by cells cultured in media including soy hydrolysate with an ornithine concentration of 2.0 mg / L or less produce higher qual...

example 3

ein Production Titer

[0123]16 soy hydrolysate lots were tested for their ability to affect the metabolomics of CHO cell production of rilonacept. Approximately 426 soy hydrolysate analytes were measured and compared to final glycoprotein titer and lactate metabolism. FIG. 5 depicts the loading plots of correlations between the soy hydrolysate analytes and maximum lactate and final glycoprotein titer. The determinations of lactate and glycoprotein titer demonstrate a negative correlation of ornithine in the soy hydrolysate.

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Abstract

The instant application provides a method for screening batches of soy hydrolysate for a desired amount of a component thereof, such as ornithine or putrescine, and selecting only those batches of soy hydrolysate that have a desired amount of such component. The present disclosure also sets forth methods for culturing cells in media supplemented with selected batches of soy to produce more consistent, high quality lots of a protein of interest. Further, the present disclosure provides a plurality of protein preparations that have each been produced by culturing cells in media supplemented with separate batches of soy hydrolysate containing a desired amount of ornithine or putrescine, whereby each batch of protein produced exhibits improved quality of the protein of interest or amount of quality protein produced.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to and the benefit of U.S. Provisional Patent Application Ser. No. 62 / 529,471, filed Jul. 6, 2017, and 62 / 625,744, filed Feb. 2, 2018. The entire contents of each above-mentioned applications are herein incorporated by reference in their entireties.INCORPORATION OF THE SEQUENCE LISTING[0002]The contents of the text filed named “REGE009P02US_SeqList.txt”, which was created on Feb. 1, 2018 and is 11.2 KB in size, are hereby incorporated by reference in their entirety.FIELD[0003]The invention relates to methods for the culturing of cells and for the production of recombinant proteins. The invention specifically relates to methods for the culturing of cells in soy hydrolysate-containing media to achieve consistent production of high quality recombinant protein.BACKGROUND[0004]Cell culture media containing protein hydrolysates, such as soy hydrolysate, are commonly used in the production of recombinant proteins...

Claims

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Application Information

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IPC IPC(8): C12P21/00C07K14/715C07K14/47
CPCC12P21/005C07K2319/30C07K14/4705C07K14/7155C07K14/415C12P21/06C12N5/0018C12N5/0682C12N2500/32C12N2500/46C07K14/71C07K2319/32
InventorCHEN, JOHNLAWRENCE, SHAWNJOHNSON, AMYLONEY, THEODOREPANGULE, RAVINDRAHANG, TA-CHUNCARVER, SCOTTSCHILLING, BERNHARD
OwnerREGENERON PHARM INC