Discovery and Control of Succinimide Formation and Accumulation at Aspartic Acid Residues in The Complementarity-Determining Region of a Therapeutic Monoclonal Antibody.

PURPOSE: Succinimide formation and isomerization alter the chemical and physical properties of aspartic acid residues in a protein. Modification of aspartic acid residues within complementarity-determining regions (CDRs) of therapeutic monoclonal antibodies (mAbs) can be particularly detrimental to the efficacy of the molecule. The goal of this study was to characterize the site of succinimide accumulation in the CDR of a therapeutic mAb and understand its effects on potency. Furthermore, we aimed to mitigate succinimide accumulation through changes in formulation. METHODS: Accumulation of succinimide was identified through intact and reduced LC-MS mass measurements. A low pH peptide mapping method was used for relative quantitation and localization of succinimide formation in the CDR. Statistical modeling was used to correlate levels of succinimide with basic variants and potency measurements. RESULTS: Succinimide accumulation in Formulation A was accelerated when stored at elevated temperatures. A strong correlation between succinimide accumulation in the CDR, an increase in basic charge variants, and a decrease in potency was observed. Statistical modeling suggest that a combination of ion exchange chromatography and potency measurements can be used to predict succinimide levels in a given sample. Reformulation of the mAb to Formulation B mitigates succinimide accumulation even after extended storage at elevated temperatures. CONCLUSION: Succinimide formation in the CDR of a therapeutic mAb can have a strong negative impact on potency of the molecule. We demonstrate that thorough characterization of the molecule by LC-MS, ion exchange chromatography, and potency measurements can facilitate changes in formulation that mitigate succinimide formation and the corresponding detrimental changes in potency.

Development of a robust functional cell-based assay for replacing the rabbit blood sugar bioidentity test of insulin glargine drug substance.

A rabbit blood sugar bioidentity assay is required by the FDA to evaluate biological activity for all insulin and its analogs per USP<121> guideline. Not only are a large number of live animals used, but the rabbit blood sugar method is also highly variable and expensive. Our goal is to develop a functional cell-based assay to replace rabbit blood sugar method. An H4IIE G6P-Luc reporter assay was developed by utilizing insulin's role in regulating hepatic gluconeogenesis pathway. It is known that Glucose 6-phosphatase is a rate-limiting enzyme in the gluconeogenesis pathway, and the mRNA expression of its catalytic subunit, G6PC, is highly regulated by insulin. A G6P-Luc stable cell line in H4IIE hepatocytes was first generated by stably expressing luciferase reporter gene driven by human G6PC promoter via lentivirus technology. The cell-based assay was developed and optimized to demonstrate good dose-dependent responsiveness to insulin. We further qualified the assay with two analysts through multiple runs, and demonstrated excellent performance characteristics of linearity, accuracy, and precision. A robustness study was then conducted to define critical factors for assay performance. We compared this newly developed assay with a previously established cell-based pIR MSD assay, which measures insulin receptor phosphorylation (pIR) in HepG2 cell line using Meso-Scale Discovery (MSD) technology. The comparability study was conducted to compare the two assays using samples generated from forced degradation. This study showed high correlation between assays, and both are stability indicating. Compared with the pIR MSD assay, the G6P-Luc assay not only has a significantly lower variability in qualification studies, but also offers many other advantages, including ease of use in a quality control laboratory with fewer steps, lower cost, and does not depend on a single vendor. In conclusion, we have developed a physiologically relevant and robust functional cell-based assay that is suitable to replace rabbit blood sugar method.

Therapeutic Effects of FGF23 c-tail Fc in a Murine Preclinical Model of X-Linked Hypophosphatemia Via the Selective Modulation of Phosphate Reabsorption.

Fibroblast growth factor 23 (FGF23) is the causative factor of X-linked hypophosphatemia (XLH), a genetic disorder effecting 1:20,000 that is characterized by excessive phosphate excretion, elevated FGF23 levels and a rickets/osteomalacia phenotype. FGF23 inhibits phosphate reabsorption and suppresses 1α,25-dihydroxyvitamin D (1,25D) biosynthesis, analytes that differentially contribute to bone integrity and deleterious soft-tissue mineralization. As inhibition of ligand broadly modulates downstream targets, balancing efficacy and unwanted toxicity is difficult when targeting the FGF23 pathway. We demonstrate that a FGF23 c-tail-Fc fusion molecule selectively modulates the phosphate pathway in vivo by competitive antagonism of FGF23 binding to the FGFR/α klotho receptor complex. Repeated injection of FGF23 c-tail Fc in Hyp mice, a preclinical model of XLH, increases cell surface abundance of kidney NaPi transporters, normalizes phosphate excretion, and significantly improves bone architecture in the absence of soft-tissue mineralization. Repeated injection does not modulate either 1,25D or calcium in a physiologically relevant manner in either a wild-type or disease setting. These data suggest that bone integrity can be improved in models of XLH via the exclusive modulation of phosphate. We posit that the selective modulation of the phosphate pathway will increase the window between efficacy and safety risks, allowing increased efficacy to be achieved in the treatment of this chronic disease. © 2017 American Society for Bone and Mineral Research.

7-Phenyl-pyrido[2,3-d]pyrimidine-2,4-diamines: novel and highly selective protein tyrosine phosphatase 1B inhibitors.

High throughput screening of the Roche compound collection led to the identification of diaminopyrroloquinazoline series as a novel class of PTP1B inhibitors. Structural modification of diaminopyrroloquinazoline series resulted in pyrido[2,3-d]pyrimidine-2,4-diamine series which was further optimized to give compounds 5 and 24 as potent, selective (except T-cell phosphatase) PTP1B inhibitors with good mouse PK properties.

A selective phosphatase of regenerating liver phosphatase inhibitor suppresses tumor cell anchorage-independent growth by a novel mechanism involving p130Cas cleavage.

The phosphatase of regenerating liver (PRL) family, a unique class of oncogenic phosphatases, consists of three members: PRL-1, PRL-2, and PRL-3. Aberrant overexpression of PRL-3 has been found in multiple solid tumor types. Ectopic expression of PRLs in cells induces transformation, increases mobility and invasiveness, and forms experimental metastases in mice. We have now shown that small interfering RNA-mediated depletion of PRL expression in cancer cells results in the down-regulation of p130Cas phosphorylation and expression and prevents tumor cell anchorage-independent growth in soft agar. We have also identified a small molecule, 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one (thienopyridone), which potently and selectively inhibits all three PRLs but not other phosphatases in vitro. The thienopyridone showed significant inhibition of tumor cell anchorage-independent growth in soft agar, induction of the p130Cas cleavage, and anoikis, a type of apoptosis that can be induced by anticancer agents via disruption of cell-matrix interaction. Unlike etoposide, thienopyridone-induced p130Cas cleavage and apoptosis were not associated with increased levels of p53 and phospho-p53 (Ser(15)), a hallmark of genotoxic drug-induced p53 pathway activation. This is the first report of a potent selective PRL inhibitor that suppresses tumor cell three-dimensional growth by a novel mechanism involving p130Cas cleavage. This study reveals a new insight into the role of PRL-3 in priming tumor progression and shows that PRL may represent an attractive target for therapeutic intervention in cancer.

Identification of a novel class of orally active pyrimido[5,4-3][1,2,4]triazine-5,7-diamine-based hypoglycemic agents with protein tyrosine phosphatase inhibitory activity.

A novel series of orally active pyrimido[5,4-3][1,2,4]triazine-5,7-diamine-based hypoglycemic agents have been identified. These compounds show non-selective inhibitory properties against a panel of protein tyrosine phosphatases including PTP1B. Compounds 12 and 13 display oral glucose lowering effects in ob/ob mice.