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.
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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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