Development and validation of a noncompetitive electrochemiluminescence-based immunoassay (ECLIA) for specific determination of insulin lispro (Humalog®) in human serum to support pharmacokinetic assessments.

Despite the importance of insulin and insulin analogs as therapeutic agents for the treatment of type I and II diabetes mellitus (DM), bioanalysis to support regulatory submissions of analogs remains a challenging endeavor. In particular, quantitation of insulin lispro by immunoanalytical methods has largely been limited to assays that display a high degree of cross-reactivity to native insulin because this analog shares extensive primary sequence homology with endogenous insulin and its efficacious circulating concentrations are low. We report herein development of the first noncompetitive electrochemiluminescence-based immunoassay (ECLIA) for specific determination of insulin lispro in serum or plasma. The new sandwich ECLIA permits accurate assessment of insulin lispro pharmacokinetics without interference from endogenous insulin. Integral to the development of this specific immunoassay was establishment of a proprietary process for affinity production of an oligoclonal monospecific guinea pig antiserum to the unique subtle structural modification in insulin lispro. We specifically optimized the ECLIA to provide reliable performance for supporting pharmacokinetic assessments in the pharmacologically relevant concentration range from 50.0 to 5,000 pM with robust performance up to 100,000 pM upon dilution. We concluded the new noncompetitive ECLIA represents a useful and convenient immunoassay for accurate quantitation of insulin lispro during pharmacokinetic assessments.

Application of size-exclusion chromatography in the investigation of the in vitro stability of proinsulin and its cleaved metabolites in human serum and plasma.

To help ensure reliability of proinsulin measurements and define the optimal matrix for conducting routine bioanalysis of this prognostic biomarker, we undertook a systematic evaluation of its in vitro stability. For this study, we subjected mono-radioiodinated forms of hPI and its cleaved metabolites to size-exclusion chromatography (FPLC-SEC employing a Superdex-75 10/30 HR column) to characterize their elution profiles following incubation in human serum and plasma. We determined that intact hPI is a substrate for serine-like protease(s) that are present in human serum. Furthermore, RIA analysis of the elution profile of unlabeled peptide demonstrated that the B-C junction is cleaved preferentially. Thus, in vitro degradation of hPI represents a potential pathway for the formation of cleaved metabolites. Our findings confirmed that EDTA plasma is the preferred matrix for quantitative determination of intact hPI and its cleaved metabolites. We concluded the SEC strategy employed in this study is broadly applicable to evaluating the in vitro stability of other peptides/proteins of diagnostic or therapeutic interest.

Engineering the proteolytic specificity of activated protein C improves its pharmacological properties.

Human activated protein C (APC) is an antithrombotic, antiinflammatory serine protease that plays a central role in vascular homeostasis, and activated recombinant protein C, drotrecogin alfa (activated), has been shown to reduce mortality in patients with severe sepsis. Similar to other serine proteases, functional APC levels are regulated by the serine protease inhibitor family of proteins including alpha(1)-antitrypsin and protein C inhibitor. Using APC-substrate modeling, we designed and produced a number of derivatives with the goal of altering the proteolytic specificity of APC such that the variants exhibited resistance to inactivation by protein C inhibitor and alpha(1)-antitrypsin yet maintained their primary anticoagulant activity. Substitutions at Leu-194 were of particular interest, because they exhibited 4- to 6-fold reductions in the rate of inactivation in human plasma and substantially increased pharmacokinetic profiles compared with wild-type APC. This was achieved with minimal impairment of the anticoagulant/antithrombotic activity of APC. These data demonstrate the ability to selectively modulate substrate specificity and subsequently affect in vivo performance and suggest therapeutic opportunities for the use of protein C derivatives in disease states with elevated serine protease inhibitor levels.