Table 1.
Similarity testing plan, analytical methods and product characteristics for the structural and functional characterization.
Table 2.
Structure and purity/variant physicochemical studies of MYL-1501D and US and EU reference products.
Fig 1.
Structural characterization and comparison of MYL-1501D, US- and EU-licensed insulin glargine.
Representative overlay of chromatographic peak profile of reduced peptide mass fingerprinting from MYL-1501D and reference products. Fragments 1–6 are described in Table 1. DTT, dithiothreitol.
Fig 2.
Representative overlay of MYL-1501D, US- and EU-licensed insulin glargine.
(A) FTIR Profiles for Secondary Structure of and (B) CD Profiles for Secondary and Tertiary Structures. CD, circular dichroism; FTIR, Fourier-transformed infrared.
Fig 3.
Representative overlay of MYL-1501D, US- and EU-licensed insulin glargine.
(A) Intrinsic Fluorescence Spectral Plots (B) Superposition of MYL-1501D X-Ray Structure and Reference Products With Insulin Glargine (4IYD) as the Base. (C) Differential Scanning Calorimetry Profiles of MYL-1501D (Red), US-Licensed Insulin Glargine (Green), and EU-Licensed Insulin Glargine (Blue).
Fig 4.
Reverse-phase high-performance liquid chromatography overlay of MYL-1501D, US- and EU-licensed insulin glargine.
MYL-1501D (Red), US-Licensed Insulin Glargine (Blue, Vial; Green, Cartridge), and EU-Licensed Insulin Glargine (Magenta).
Fig 5.
Scatter plot distributions of binding kinetic constants of MYL-1501D, US- and EU-licensed insulin glargine.
Binding Kinetic Constants i.e., ka, kd, and KD for (A-C) IR-A (Short-Form), (D-F) IR-B (Long-Form), and (G-I) IGF-1R for MYL-1501D, US-Licensed Insulin Glargine, and EU-Licensed Insulin Glargine. Multiple lots of MYL-1501D, US-, and EU-licensed insulin glargine were analyzed, and data are expressed as mean ± 95% CI. Each dot represents one lot for which data were collected from 3 independent runs, and mean is presented. Mean ± 3 SD range derived from innovator product is presented as solid green line for US-licensed insulin glargine and dotted blue line for EU-licensed insulin glargine. *P<0.05, **P<0.01, and ***P<0.001 in TOST/Equivalence test. IGF-1R, insulin growth factor-1 receptor; IR, insulin receptor; ka, association rate constant; kd, dissociation rate constant; KD, equilibrium dissociation constant; TOST, Two One-Sided T-test.
Fig 6.
Representative binding response graphs of the insulin receptor-A (short-form) kinetic binding assay.
(A) MYL-1501D, (B) US-Licensed Insulin Glargine, and (C) EU-Licensed Insulin Glargine. Each lot was analyzed over 6 concentrations, and kinetic parameters were evaluated by 1:1 interaction model using global fit. Each lot was analyzed 3 independent times, and mean KD value was reported. KD, equilibrium dissociation constant.
Fig 7.
Representative binding response graphs of the insulin receptor-B (long-form) kinetic binding assay.
(A) MYL-1501D, (B) US-Licensed Insulin Glargine, and (C) EU-Licensed Insulin Glargine. Each lot was analyzed over 6 concentrations, and kinetic parameters were evaluated by 1:1 interaction model using global fit. Each lot was analyzed 3 independent times, and mean KD value was reported. KD, equilibrium dissociation constant.
Fig 8.
Representative binding response graph of insulin-like growth factor receptor-1 binding kinetics assay.
(A) MYL-1501D, (B) US-Licensed Insulin Glargine, and (C) EU-Licensed Insulin Glargine. Each lot was analyzed over 6 concentrations, and kinetic parameters were evaluated by 1:1 interaction model using global fit. Each lot was analyzed 3 independent times, and mean KD value was reported. KD, equilibrium dissociation constant.
Table 3.
Insulin receptor IR-A, IR-B and IGF-1R, binding kinetics of MYL-1501D, US- and EU-licensed insulin glargine.
Fig 9.
Representative dose-response curves of insulin receptor-A (short-form) phosphorylation assay.
Recombinant CHO-K1 Cells Expressing Insulin Receptor-A were used in the assay. (A) MYL-1501D, (B) US-Licensed Insulin Glargine, and (C) EU-Licensed Insulin Glargine. All samples were analyzed against a common insulin glargine reference standard (IG standard) to normalize against day-to-day variability. Each lot was analyzed using 3 independent dilution preparations on an assay plate. Mean ± SEM for the % phosphorylation response is plotted (y-axis) against the concentration (x-axis). Four parameter logistic curve fit was applied to evaluate EC50. Each sample was analyzed on 3 independent assay plates, and average is reported. CHO, Chinese hamster ovary; EC50, half maximal effective concentration.
Fig 10.
Representative dose-response curves of insulin receptor-B (long-form) phosphorylation assay.
Recombinant CHO-K1 Cells Expressing Insulin Receptor-B were used in the assay. (A) MYL-1501D, (B) US-Licensed Insulin Glargine, and (C) EU-Licensed Insulin Glargine. All samples were analyzed against a common insulin glargine reference standard (IG standard) to normalize against day-to-day variability. Each lot was analyzed using 3 independent dilution preparations on an assay plate. Mean ± SEM for the % phosphorylation response is plotted (y-axis) against the concentration (x-axis). Four parameter logistic curve fit was applied to evaluate EC50. Each sample was analyzed on 3 independent assay plates, and average is reported. CHO, Chinese hamster ovary; EC50, half maximal effective concentration.
Fig 11.
Representative dose-response curves of total insulin receptor phosphorylation assay in HepG2 cells.
(A) MYL-1501D, (B) US-Licensed Insulin Glargine, and (C) EU-Licensed Insulin Glargine. All samples were analyzed against a common insulin glargine reference standard (IG standard) to normalize against day-to-day variability. Each lot was analyzed using 3 independent dilution preparations on an assay plate. Mean ± SEM for the % phosphorylation response is plotted (y-axis) against the concentration (x-axis). Four parameter logistic curve fit was applied to evaluate EC50. Each sample was analyzed on 3 independent assay plates, and average is reported. CHO, Chinese hamster ovary; EC50, half maximal effective concentration.
Fig 12.
Scatter plot distribution of average relative potency of MYL-1501D, US- and EU-licensed insulin glargine.
(A) IR Phosphorylation in HepG2 Cells, (B) IR-A Phosphorylation in CHO-K1 Cells Expressing IR-A, (C) IR-B Phosphorylation in CHO-K1 Cells Expressing IR-B, (D) Glucose Uptake Activity in 3T3-L1 Cells, (E) Adipogenicity Activity in 3T3-L1 Cells, (F) Inhibition of Stimulated Lipolysis Activity in 3T3-L1 Cells, and (G) Mitogenic Activity by Saos-2 Cells. Multiple lots of MYL-1501D, US-, and EU-licensed insulin glargine were analyzed, and data are expressed as mean ± 95% CI. Each dot represents one lot for which data were collected from 3 independent runs, and mean is presented. Mean ± 3 SD range derived from innovator product is presented as solid green line for US-licensed insulin glargine and dotted blue line for EU-licensed insulin glargine. *P<0.05, **P<0.01, and ***P<0.001 in TOST/Equivalence test. CHO, Chinese hamster ovary; GOPOD, glucose oxidase/peroxidase; IR, insulin receptor; Saos-2, sarcoma osteogenic cell; TOST, Two One-Sided T-test.
Fig 13.
Representative dose-response curves of enzymatic glucose uptake assay in 3T3-L1 cells.
(A) MYL-1501D, (B) US-Licensed Insulin Glargine, and (C) EU-Licensed Insulin Glargine. All samples were analyzed against a common insulin glargine reference standard (IG standard) to normalize against day-to-day variability. Each lot was analyzed using 3 independent dilution preparations on an assay plate. Mean ± SEM for the % glucose remaining in the media is plotted (y-axis) against the concentration (x-axis). Four parameter logistic curve fit was applied to evaluate EC50. Each sample was analyzed on 3 independent assay plates, and average is reported. EC50, half maximal effective concentration.
Fig 14.
Representative dose-response curves of adipogenesis assay in 3T3-L1 cells.
(A) MYL-1501D, (B) US-Licensed Insulin Glargine, and (C) EU-Licensed Insulin Glargine. All samples were analyzed against a common insulin glargine reference standard (IG standard) to normalize against day-to-day variability. Each lot was analyzed using 3 independent dilution preparations on an assay plate. Mean ± SEM for the % triglycerides measured is plotted (y-axis) against the concentration (x-axis). Four parameter logistic curve fit was applied to evaluate EC50. Each sample was analyzed on 3 independent assay plates, and average is reported. EC50, half maximal effective concentration.
Fig 15.
Representative dose-response curves graph of inhibition of stimulated lipolysis assay in 3T3-L1 cells.
(A) MYL-1501D, (B) US-Licensed Insulin Glargine, and (C) EU-Licensed Insulin Glargine. All samples were analyzed against a common insulin glargine reference standard (IG standard) to normalize against day-to-day variability. Each lot was analyzed using 3 independent dilution preparations on an assay plate. Mean ± SEM for the % glycerol measured is plotted (y-axis) against the concentration (x-axis). Four parameter logistic curve fit was applied to evaluate EC50. Each sample was analyzed on 3 independent assay plates, and average is reported. EC50, half maximal effective concentration.
Fig 16.
Representative dose-response curves of mitogenic assay in Saos-2 cells.
(A) MYL-1501D, (B) US-Licensed Insulin Glargine, and (C) EU-Licensed Insulin Glargine. All samples were analyzed against a common insulin glargine reference standard (IG standard) to normalize against day-to-day variability. Each lot was analyzed using 3 independent dilution preparations on an assay plate. Mean ± SEM for the % proliferation is plotted (y-axis) against the concentration (x-axis). Four parameter logistic curve fit was applied to evaluate EC50. Each sample was analyzed on 3 independent assay plates, and average is reported. EC50, half maximal effective concentration; Saos-2, sarcoma osteogenic cell.
Fig 17.
Scatter plot distribution of in-vivo rabbit bioassay activity for MYL-1501D, US-, and EU-licensed insulin glargine.
Nine lots of MYL-1501D and 6 lots each of US- and EU-licensed insulin glargine were analyzed, and data are expressed as mean ± 95% CI. Mean ± 3 SD range derived from innovator product is presented as solid green line for US-licensed insulin glargine and dotted blue line for EU-licensed insulin glargine. *P<0.05, **P<0.01, and ***P<0.001 in TOST/Equivalence test. Potency of the 2 USP reference standards (USP-RS) used in the study are presented as black symbols (▲ USP human insulin standard lot JOJ250, ▼ USP insulin glargine standard lot F009M0). Limit of the biopotency value, ie, ≥15 USP units/mg, as per USP <121> is presented as red dotted line. TOST, Two One-Sided T-test; USP, US Pharmacopeia.