Design and preparation of chimeric hyaluronidase as a chaperone for the subcutaneous administration of biopharmaceuticals

• Novel fused hyaluronidase rhPH20-HSA and rhPH20-Fc were designed and expressed.
• Cell culture and purification process for novel hyaluronidase were developed.
• Long-acting hyaluronidase extended the channels opening in subcutaneous space.
• The improvement by rhPH20-Fc for 150 kDa Stelara was not significant over rhPH20.
• For TFI with molecular weight 250 kDa, PH20-Fc performed much better than rhPH20.

Subcutaneous (SC) delivery of biomacromolecular pharmaceuticals such as proteins often encounter barriers in the extracellular matrix, especially the hyaluronan (HA) network. In this study, chimeric hyaluronidases were designed, prepared and tested for assisting biopharmaceuticals in ID administration in mice as replacement of SC administration. The chimeras were hyaluronidase (rhPH20) conjugated with human serum albumin (rhPH20-HSA) and antibody Fc fragment (rhPH20-Fc). Expression of the new protein was undertaken in CHO cells cultured in a 5-L disposable bioreactor. Purification was carried out by a series of chromatographic methods to obtain high-purity products of 61 kDa (rhPH20), 79 kDa (rhPH20-HSA) and 190 kDa (rhPH20-Fc). The chimeric proteins rhPH20-HSA and rhPH20-Fc performed fairly well as spreading factors in short-term trypan blue intradermal (ID) infusion in comparison with recombinant hyaluronidase (rhPH20). They extended the channel opening from 24 h (rhPH20) to 85–120 h in vivo. The specific activity of rhPH20-Fc was 35,600 U/mg, higher than that of rhPH20-HSA (10,000 U/mg). Co-administration of rhPH20-Fc with two biomacromolecular pharmaceuticals, Stelara (150 KDa) and TNFRII-Fc-IL1ra (TFI, 250 kDa), through an ID route increased the bioavailability from 86% to 93% and from 64% and 97%, respectively, compared with rhPH20. The pharmacokinetic profile of ID administrated larger TFI was significantly improved through cooperation with the long-acting hyaluronidase.