Establishment Of Cell Lines To Produce Recombinant Protein With High-mannose-type N Glycan Structures

Therapeutic proteins are an important developing part of the modern biopharmaceutical industry,providing novel therapies to intractable diseases including cancers and autoimmune diseases.Comparing to the traditional small molecule drugs,therapeutic proteins own remarkable advantages including higher specificity,lower toxicity and longer half-life in human internal circulation.The protein expression system,or the host cell is a key factor that determine the pharmacokinetic and pharmacology properties of therapeutic proteins.The human embryonic kidney 293(HEK293)cell line has been widely used to produce recombinant proteins in both basic science and industry,since this cell line does not express any immunogenic glycan structure like Gal-al,3-Gal and Neu5Gc in the CHO cell line.The heterogeneity of glycan structures is one of the most challenging issues in the production of therapeutic proteins.Previously,we knocked out genes encoding ?1,2-mannosidase-Is,MAN1A1,MAN1A2,and MAN1B1,in HEK293 cells,establishing a triple-knockout(T-KO)cell line,which produced recombinant protein with mainly high-mannose-type N-glycans.Here,we further knocked out MANIC1 and MGAT1 encoding another Golgi a1,2-mannosidase-I and N-acetylglucosaminyltransferase-1,respectively,based on the T-KO cells.Two recombinant proteins,lysosomal acid lipase(LIPA)and immunoglobulin G1(1gG1),were expressed in the quadruple-KO(QD-KO)and quintuple-KO(QT-KO)cell lines.Glycan structural analysis revealed that all hybrid-type and complex-type N-glycans were eliminated,and only the high-mannose-type N-glycans were detected among the recombinant proteins prepared from the QD-KO and QT-KO cells.Overexpression of the oncogenes MYC and&MYCN recovered the slow growth in QD-KO and QT-KO without changing the glycan structures.Our results suggest that these cell lines could be suitable platforms to produce homogeneous therapeutic proteins.