Modulation of ornithine metabolism to manipulate high mannose glycoform content of recombinant proteins

A recombinant protein and high mannose technology, which is applied in immunoglobulin, animal/human protein, tissue culture, etc., can solve problems affecting the therapeutic efficacy of recombinant protein drugs, etc.

Active Publication Date: 2020-10-27
AMGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Glycosylation affects the therapeutic efficacy of recombinant protein drugs

Method used

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  • Modulation of ornithine metabolism to manipulate high mannose glycoform content of recombinant proteins
  • Modulation of ornithine metabolism to manipulate high mannose glycoform content of recombinant proteins
  • Modulation of ornithine metabolism to manipulate high mannose glycoform content of recombinant proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Extracellular ornithine levels were found to correlate with high mannose glycoform content. Eight CHO cell lines expressing recombinant antibodies with high mannose glycoform content ranging from 20% were selected for this experiment (cell line A-cell line H). Cells were grown in fed-batch culture for 10 days in shake flasks using two different proprietary cell culture media (Medium #1 and Medium #2), each without ornithine. Spent medium samples were removed on days 8, 9, and 10 of culture and subjected to large-scale metabolomics analysis. %HM was determined using the Endo-H rCE-SDS method followed by replacement by the HILIC method described below. Determination of relative levels of ornithine in spent media by large-scale metabolomics analysis in which media components were separated by liquid chromatography and detected by high-resolution spectrometry . Components are identified by matching fragment spectra to a spectral library of known compounds. The relative ...

Embodiment 2

[0131] Arginase 1 mRNA expression levels were determined on selected days during the 10-day fed-batch cultures using the eight cell lines described in Example 1.

[0132] mRNA expression levels were assessed using the QuantiGene Multiplex Assay Kit (Affymetrix, Inc., Santa Clara, CA) according to the manufacturer's instructions.

[0133] Arginase 1 (an enzyme that catalyzes the conversion of arginine to ornithine) was found to be upregulated in a time course-dependent manner in cell lines with higher levels of high mannose, see Figure 4 . This suggests that specific targeting with arginase to block arginase activity and reduce the amount of ornithine produced can be used to reduce high mannan levels.

Embodiment 3

[0135] This example demonstrates that manipulation of the high mannose glycoform content of recombinant glycoproteins is addressed by modulating ornithine accumulation in host cells expressing the recombinant glycoproteins.

[0136] Cell Lines, Cell Culture and Media

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Abstract

The present invention relates to a method for manipulating the high mannose glycoform content of recombinant glycoproteins by modulating ornithine metabolism during cell culture.

Description

[0001] This application claims the benefit of US Provisional Application No. 61 / 926,481 filed January 13, 2014, which is hereby incorporated by reference in its entirety. [0002] Background of the invention [0003] Higher eukaryotes carry out a variety of post-translational modifications, including methylation, sulfation, phosphorylation, addition of lipids, and glycosylation. Many of the secreted proteins, membrane proteins and proteins targeted to vesicles and certain intracellular organelles are known to be glycosylated. Glycosylation, the covalent attachment of sugar moieties to specific amino acids, is one of the most common but important post-translational modifications of recombinant proteins. Protein glycosylation has multiple functions in cells, including its essential role in protein folding and quality control, molecular trafficking and sorting, and cell surface receptor interactions. [0004] N-linked glycosylation involves the addition of oligosaccharides to asp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/00C07K16/00C12N1/38C12N5/00
CPCC07K16/00C12N1/38C12N2510/02C07K2317/14C07K2317/41C12P21/005C07K14/52C12N2500/32C12N2500/33C12N2500/46C12N2500/90C12N2500/99C12N2501/70C12N2501/72C12N2501/73C12N2501/999C12N2521/00C12N5/0682
Inventor S.康C.小黄H.巴霍尔达里安P.邦达伦科Z.张
Owner AMGEN INC
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