(M1030-09-57) Measuring and Controlling Potency of a Targetless Biologic: Study of PEGylated Hemoglobin

Purpose: As per the FDA, a potency assay is required due to the complexity and heterogeneity of biologics. Hemoglobin based oxygen carriers have been under development to treat ischemic conditions by delivering oxygen to hypoxic tissues. This class of molecules poses a unique challenge due to lack of any target receptor. Here we present a case study of PP-007 (PEGylated carboxy hemoglobin bovine) where a unique activity assay was utilized to quantify oxygen affinity and gas transfer activity of PP-007. Moreover, we demonstrate how we were able to target hypoxia by adjusting the oxygen affinity of PP-007 by controlling the degree of PEGylation. Activity of the molecule was measured over refrigerated stability for up to 36M. Clinical safety of the molecule was established in Phase Ib trial in acute Ischemic Stroke population undergoing mechanical thrombactomy.

Methods: To quantify oxygen affinity of PP-007, P50 attribute (PO2 at which 50% molecules are oxygenated) was selected. A clinical Hemox (blood gas analyzer) instrument was used to generate deoxygenation/oxygenation dissociation curve by varying pO2 in a cuvette from 0 to 150 mm Hg. P50 along with hill coefficient was derived from the dissociation curve. Purified bovine hemoglobin was used as standard to confirm suitability of the measurement. To generate PP-007, multi-PEGylation was carried out to left shift p50 of the hemoglobin i.e. higher affinity for oxygen. Multi-PEGylated hemoglobin was generated by amine directed 5k-Sc PEGylation of purified bovine hemoglobin in pH 8.0 phosphate buffer. A library of PEGylated hemoglobin was created by varying PEG to protein ratio. Concentration of the PEG in the reaction was varied from 12.5 g/L to 125.0 g/L. Degree of PEGylation was measured by SDS-PAGE/CE-SDS. Gas transfer ability was measured by incubating PP-007 samples in presence of air as well as hypoxia and measuring exchange of CO with O2 (i.e. conversion of %HbCO to %HbO2).

Results: Bovine hemoglobin was found to have oxygen affinity (p50) similar to that of human hemoglobin (~25 mm Hg). Based on the dissociation curve, PEGylation resulted increased oxygen affinity (p50 of 10) but reduced cooperativity for tetrameric structure. This p50 would result in release of oxygen to hypoxic tissues with low PO2 that will not be supplied by red blood cells. P50 method was qualified for release and stability testing. There was inverse relationship between degree of PEGylation and p50 with no further drop in p50 beyond 10 PEG. PP-007 with an average of 9 molecules of PEG per molecule of hemoglobin was chosen for further gas transfer studies. PP-007 in its HbCO form did not dissociate into DeoxyHb under hypoxic (N2) environment, which indicated and supported long term stability of the PP-007. The results were confirmed by analysis of samples aged up to 36M. Phase Ib safety trial in Acute Ischemic stroke population showed no safety concerns.

Conclusion: Here, we have engineered an oxygen carrier that can specifically deliver oxygen to hypoxic tissues by virtue of lower P50. An in vitro oxygen binding and affinity-based potency assay was qualified for release and stability testing. A gas exchange assay was developed for orthogonal assessment of functionality in vitro. Interestingly, in its HbCO form showed no loss of CO under hypoxic conditions which was utilized for designing argon layered secondary packaging for long term storage.

Impact of degree of PEGylation on p50 of PP-007 showing inverse relationship with no improvement beyond 10 PEG.
Gas transfer assay. A) under hypoxic environment, no remarkable change in HbCO is seen. B) Under oxygenated environment, a concentration dependent exchange was observed. [RHB=HHB=DeoxyHb, MetHb=Oxidized hemoglobin]