Aggregation of human plasma and of human blood induced in vitro by pegfilgrastim originator formulation buffer and pegfilgrastim products

PEGylated recombinant human granulocyte colony stimulating factor (pegfilgrastim) is used clinically to reduce the incidence and duration of severe neutropenia in patients who have received chemotherapy treatment. Pegfilgrastim products are administered by subcutaneous injection. We herein report that solutions of pegfil-grastim originator product Neulasta (R), of a biosimilar product candidate, and also of the pegfilgrastim originator formulation buffer, induced aggregate formation when mixed in vitro with human plasma, and formation of large membranous aggregated structures when mixed with human blood. Human donor variability in the plasma aggregation induced by pegfilgrastim products was observed. In all donors less aggregation occurred in plasma mixtures with the biosimilar pegfilgrastim product candidate compared to the originator products. Instantaneous aggregation of erythrocytes and formation of large membranous aggregated structures of erythrocytes occurred in mixtures of human blood with pegfilgrastim buffer or pegfilgrastim products. The formation of the large membranous aggregated structures likely involved fusion of erythrocyte membranes; erythrocyte membrane fusion events were observed. Pegfilgrastim proteins in the products accelerated the formation of irreversible erythrocyte aggregated structures. Pegfilgrastim originator formulation buffer (10 mM Na-acetate pH 4.0, 274 mM sorbitol, 0.004 % polysorbate 20) was identified as the main driver of the plasma and erythrocyte aggre-gation. Lipoprotein aggregation at low pH in the presence of sorbitol and erythrocyte membrane fusion induced by the lipoprotein aggregates, are proposed as the main mechanisms for the formation of plasma and blood aggregates. Such aggregation phenomena may also occur during pegfilgrastim clinical use and may be related to known side effects and individual variability in the efficacy of pegfilgrastim therapy.