Structure And Functional Characterization Of RHuPH20 Produced In CHO Cells

PurposeMonoclonal antibody-based treatment has been established as one of the most succedssful therapeutic strategies with the rapid development of biotechnology.Dosing of therapeutic antibodies is often very high,many therapies administered by IV infusion.This brings inconvenience to the patients and increases the cost of medical care.Subcutaneous(SC)administration of m Abs has been shown to reduce administration times and health-care costs relative to IV infusion.However,this delivery method is challenged by limitations on drug volume.With the development of technology,the co-formulation with recombinant human hyaluronidase(r Hu PH20)makes the relatively pain-free administration of m Abs.r Hu PH20 can temporarily increase the subcutaneous space through the degradation of extracellular matrix.The recombinant human hyaluronidase(r Hu PH20)was expressed by Chinese Hamster Ovary Cells(CHO cells).Then we combine the high performance liquid chromatography with mass spectrometry for detailed identification and characterization of the recombinant human hyaluronidase(r Hu PH20).Then the in vitro and vivo biological activities were assessed,and in vivo pharmacokinetic analysis of an anti-PD-L1 antibody r Hu PH20 co-formulation was also carried out.Finally,an in-depth characterization of r Hu PH20 was carried out,N-glycans were identified and 6 occupied N-glycosylation sites were pinpointed for the first time.This study offers the structural basis for the next phase of animal experiments and initial clinical application.Methods? SDS-PAGE analysis.? Intact protein LC/MS analysis? Peptide level glycan analysis? Cleaved glycan level analysis? In vitro biological analysis using turdimimetric method.? In vivo biological analysis by interstitial dispersion experiments(trypan blue),and pharmacokinetic analysis of an anti-PD-L1 antibody r Hu PH20 co-formulation.Results? SDS-PAGE analysis showed that the actual MW was excellent agreement with theoretical data.? After PNGase F digestion,the mass of r Hu PH20 main peak is 52261.75 Da.? Peptide level glycan analysis showed that 6 occupied N-glycosylation sites were pinpointed: Asn 47,131,200,219,333 and 358 respectively.? Cleaved glycan level analysis confirmed there were 18 different glycan structures in r Hu PH20,which were mainly composed of fucosylated glycan with two antenna and high-mannose glycans.? In vitro and in vivo studies showed that the bioactivity of r Hu PH20 decreased dramatically after PNGase F digestion.