Nucleic acid constructs, vectors, and methods for reducing the high mannose content of recombinant proteins

Abstract

This disclosure provides a nucleic acid construct comprising a first polynucleotide encoding the MGAT1 gene or a homologous gene having at least 85% sequence identity with it, a second polynucleotide encoding the MGAT2 gene or a homologous gene having at least 85% sequence identity with it, and a third polynucleotide encoding the MAN2A2 gene or a homologous gene having at least 85% sequence identity with it. This disclosure also provides a combination of nucleic acid constructs comprising the first, second, and third nucleic acid constructs, each comprising a first polynucleotide encoding the MGAT1 gene or a homologous gene having at least 85% sequence identity with it, a second polynucleotide encoding the MGAT2 gene or a homologous gene having at least 85% sequence identity with it, and a third polynucleotide encoding the MAN2A2 gene or a homologous gene having at least 85% sequence identity with it. A method for regulating high mannose glycoform content of recombinant proteins during mammalian cell culture is also provided, comprising expressing the recombinant protein and overexpressing three proteins involved in the N-glycosylation pathway in mammalian host cells. Therefore, this disclosure provides an efficient method for reducing high mannose glycoform content of recombinant proteins in CHO cells.

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