LARIX BIOSCIENCE LLC — Department of Health and Human Services SBIR Phase I: 300

Description

Human Glyco engineered CHO Cells Abstract Glycosylation is well known to modulate the activity of therapeutic proteins Recently sialylation of the N linked glycan chains has been shown to significantly affect recombinant glycoprotein stability and activity For example the degree of sialylation directly correlates with serum half life Sialic acids are also important modulators of immunogenicity a major problem with protein based therapeutics Immunogenicity inversely correlates with the degree of sialylation Negatively charged sialic acids influence protein specific parameters like thermal stability resistance to proteolysis and solubility Despite the importance of sialylation sialic acid incorporation by the production platforms in wide use today including Chinese hamster ovary CHO cells is inefficient and highly variable a product consistency issue Variable sialylation is due largely to the activity of sialidases as is the incorporation of the non human sialic acid Neu Gc to which most humans possess antibodies Therefore it is of great interest to develop methods or platforms to yield uniformly sialylated proteins with a fully human sialylation profile While several methods have been described to address sialic acid incorporation none has yet been widely adopted due to inefficiencies or the increased cost of production We have recently modified the CRISPR Cas system for genome engineering to develop a method for rapid efficient generation of homozygous negative cell lines Double allele Knockout DAKO technology We will apply DAKO to delete sialidase and cytidine monophosphate N acetylneuraminic acid hydroxylase CMAH in CHO cells Antibodies and other glycoproteins produced in these cells will have a high degree of sialic acid incorporation and will be free of Neu Gc Narrative Therapeutic proteins biologics such as antibodies address critical health care needs that are currently unmet by small molecule drugs To minimize adverse immune reactions immunogenicity and to maintain a high degree of functionality a human pattern of glycosylation is desirable with a high degree of sialic acid incorporation We have designed a superior recombinant protein production platform that will yield purely human glycosylation including a high degree of sialic acid incorporation without contamination with nonhuman sialic acids