Glargine insulin loaded lipid nanoparticles: Oral delivery of liquid and solid oral dosage forms.

BACKGROUND AND AIMS: The oral administration of insulin has so far been precluded by gastro-intestinal enzyme degradation and poor intestinal absorption. Preliminary evidence for peptide uptake by the gut has recently been obtained, by our research group, following the administration of nanostructured lipid-carrier suspensions loaded with glargine insulin in healthy animal models. METHODS AND RESULTS: In this experimental study, glargine insulin-loaded nanostructured lipid carriers have been converted into solid oral dosage forms (tablets, capsules), that are more suitable for administration in humans and have prolonged shelf-life. The liquid and solid oral dosage forms were tested for glargine insulin uptake and glucose responsivity in healthy and streptozotocin-induced diabetic rats (6 animals in each group). A suitable composition gave redispersible solid oral dosage forms from glargine insulin-loaded carriers, using both spray-drying and freeze-drying. It was observed that the liquid and solid formulations had relevant hypoglycaemic effects in healthy rats, while only capsules were efficacious in diabetic rats; probably because of gut alterations in these animal models. Detected glargine insulinaemia was consistent with a glycaemic profile. CONCLUSION: The formulations under study showed their potential as oral glucose-lowering agents, particularly when used as capsules. However, further study is needed to produce a useful orally-active insulin preparation.

NMR Spectroscopy for Protein Higher Order Structure Similarity Assessment in Formulated Drug Products.

Peptide and protein drug molecules fold into higher order structures (HOS) in formulation and these folded structures are often critical for drug efficacy and safety. Generic or biosimilar drug products (DPs) need to show similar HOS to the reference product. The solution NMR spectroscopy is a non-invasive, chemically and structurally specific analytical method that is ideal for characterizing protein therapeutics in formulation. However, only limited NMR studies have been performed directly on marketed DPs and questions remain on how to quantitively define similarity. Here, NMR spectra were collected on marketed peptide and protein DPs, including calcitonin-salmon, liraglutide, teriparatide, exenatide, insulin glargine and rituximab. The 1D 1H spectral pattern readily revealed protein HOS heterogeneity, exchange and oligomerization in the different formulations. Principal component analysis (PCA) applied to two rituximab DPs showed consistent results with the previously demonstrated similarity metrics of Mahalanobis distance (DM) of 3.3. The 2D 1H-13C HSQC spectral comparison of insulin glargine DPs provided similarity metrics for chemical shift difference (Δδ) and methyl peak profile, i.e., 4 ppb for 1H, 15 ppb for 13C and 98% peaks with equivalent peak height. Finally, 2D 1H-15N sofast HMQC was demonstrated as a sensitive method for comparison of small protein HOS. The application of NMR procedures and chemometric analysis on therapeutic proteins offer quantitative similarity assessments of DPs with practically achievable similarity metrics.

Formulary Considerations for Insulins Approved Through the 505(b)(2) "Follow-on" Pathway.

OBJECTIVE: To summarize formulary-relevant issues for follow-on insulins approved through the Food and Drug Administration (FDA) 505(b)(2) approval pathway (Basaglar and Admelog). DATA SOURCES: A search of the MEDLINE database was performed for articles pertaining to clinical and formulary considerations for follow-on insulin products through July 2018. STUDY SELECTION AND DATA EXTRACTION: All clinical trials used in the 505(b)(2) approval process for follow-on insulin glargine and insulin lispro products were included and summarized. DATA SYNTHESIS: Follow-on insulin glargine and insulin lispro products have been recently approved as the first lower-cost alternatives to innovator insulin products. The follow-on insulins were approved via the 505(b)(2) pathway, making them neither generics nor biosimilars. Current data do not suggest any clinically relevant differences between the follow-on insulins and their respective innovator products. Clinicians should be aware that follow-on insulins will be reclassified as biologic products in the year 2020. Relevance to Patient Care and Clinical Practice: This article provides information about currently available follow-on insulin products that were approved through the 505(b)(2) pathway, including product characteristics and efficacy and safety data. These products will likely be considered for both clinical use and formulary placement because of their potentially lower cost compared with innovator products. CONCLUSIONS: Follow-on insulin products approved through the 505(b)(2) pathway are supported by robust efficacy and safety data. As new follow-on insulins are approved and the regulatory change that will occur with these products in 2020 approaches, formulary decisions and clinical policies (eg, substitution) will continue to be revisited.

Physico-chemical stability of co-formulation of PEGylated human amylin with insulin.

Since the discovery of amylin no combined formulation with insulin has been made available. Amylin or its triple proline analog pramlintide are not compatible in solution with insulin. The drug candidate hAmy-PEG5k is a novel monoPEGylated amylin derivative with improved physicochemical properties and retained similar pharmacological activity compared to free amylin and pramlintide. We have investigated the short- and long-term physicochemical compatibility of hAmy-PEG5k co-formulated with slow-acting human insulin analogs glargine or detemir. While human amylin promptly aggregates over a large range of pH, and both free and in the presence of regular, glargine or detemir insulin, the hAmy-PEG5k analog is stable at these conditions as shown by Thioflavin T (ThT) binding assay. When hAmy-PEG5k (100 or 500 µg/mL) was added to the commercial formulations of either insulin glargine or detemir (95 IU/mL), the combinations remained stable after 6 months stored at 4 °C, as probed by ThT, dynamic light scattering (DLS) measurements and high performance liquid chromatography (HPLC) analyses, confirming the absence of amyloid fibers, minor aggregation products or loss of material. These results suggest hAmy-PEG5k and the insulin analogs glargine and detemir are physicochemically compatible and are candidate ready-to-use fixed-dose combinations.

Non-metabolisable insulin glargine does not promote breast cancer growth in a mouse model of type 2 diabetes.

AIMS/HYPOTHESIS: Previous epidemiological studies have reported a potential link between insulin analogues and breast cancer; however, a prospective randomised controlled trial showed neutral effects of insulin glargine on cancer risk. Insulin glargine is metabolised in vivo to an M1 metabolite. A question remains whether a subset of individuals with slower rates of glargine metabolism or who are on high doses could, theoretically, have an increased risk of cancer progression if a tumour is already present. In this study, we aimed to determine whether a non-metabolisable form of insulin glargine induced murine breast cancer growth. METHODS: A mouse model of type 2 diabetes (MKR) was used for these studies. MKR mice were injected with two murine mammary cancer cell lines: Mvt-1 cells (derived from MMTV-c-Myc/Vegf tumours) and Met1 cells (derived from MMTV-polyoma virus middle T antigen tumours). Mice were treated with 25 U/kg per day of the long-acting insulin analogues, insulin glargine, insulin detemir, insulin degludec or non-metabolisable glargine, or vehicle. RESULTS: No difference in tumour growth was seen in terms of tumour size after insulin glargine, detemir, degludec or vehicle injections. Non-metabolisable glargine did not increase tumour growth compared with insulin glargine or vehicle. Insulin glargine and non-metabolisable glargine led to insulin receptor phosphorylation in vivo rather than IGF-1 receptor phosphorylation. CONCLUSIONS/INTERPRETATION: These results demonstrate that in a mouse model of type 2 diabetes, at high concentrations, basal insulin analogues and a non-metabolisable glargine analogue do not promote the progression of breast tumours.

Replacement of the CysA7-CysB7 disulfide bond with a 1,2,3-triazole linker causes unfolding in insulin glargine.

Two analogues of insulin glargine containing a 1,4-disubstituted 1,2,3-triazole group in place of the CysA7-CysB7 disulfide bond were prepared using CuAAC click chemistry to efficiently join the peptide chains. The resulting insulin analogues were analysed by circular dichroism spectroscopy to assess whether this modification compromised the folding pattern of the native form. Investigations, including an in vivo murine study, revealed that these analogues were not biologically active and that the structures were significantly unfolded, an outcome which suggests that maintaining a precise inter-chain distance is critical to the structure of the insulin hormone.

Sensitive and label-free SPR biosensing platforms for high-throughput screening of plasma membrane receptors interactions with insulin-like targets of hypoglycaemic activity.

Progress in medical sciences aims for tailored therapy of civilization diseases like diabetes. Preclinical screening of new medicines superior to insulin should include the verification of their affinity to the membrane receptors naturally stimulated by this hormone: insulin receptor isoforms A and B and insulin-like growth factor receptor. Considering that the affinity constants obtained using different experimental conditions are incomparable, it is essential to develop a robust and reliable method to analyze these interactions. The versatile SPR platform developed in this study enables the evaluation of the bioactivity of hypoglycaemic molecules. Thanks to the comprehensive characterization of miscellaneous aspects of the analytical platform, including the design of the SPR biosensor receptor layer, ensuring interaction specificity, as well as the quality control of the standards used (human insulin, HI; long-acting insulin analog: glargine, Gla), the feasibility of the method of equilibrium and kinetic constants determination for insulin-like targets was confirmed. SPR assays constructed in the direct format using IR-A, IR-B, and IGF1-R receptor proteins show high sensitivities and low detection limits towards insulin and glargine detection in the range of 18.3-53.3 nM with no signs of mass transport limitations. The improved analytical performance and stability of SPR biosensors favor the acquisition of good-quality kinetic data, while preservation of receptors activity after binding to long-chain carboxymethyldextran, combined with spontaneous regeneration, results in stability and long shelf life of the biosensor, which makes it useful for label-free insulin analogs biosensing and thus extensive screening in diabetic drugs discovery.

Physicochemical and functional characterization of MYL-1501D, a proposed biosimilar to insulin glargine.

Insulin glargine is a long-acting analogue of human insulin that has been used to manage hyperglycemia in patients with diabetes mellitus (DM) for nearly 20 years. Insulin glargine has a relatively constant concentration-time profile that mimics basal levels of insulin and allows for once-daily administration. MYL-1501D is a biosimilar insulin glargine designed to offer greater access of insulin glargine to patients, with comparable efficacy and safety to the marketed reference product. We conducted a comprehensive panel of studies based on a formal analysis of critical quality attributes to characterize the structural and functional properties of MYL-1501D and reference insulin glargine products available in the United States and European Union. MYL-1501D was comprehensively shown to have high similarity to the reference products in terms of protein structure, metabolic activity (both in vitro cell-based assays and in vivo rabbit bioassays), and in vitro cell-based assays for mitogenic activity. The structural analyses demonstrated that the primary protein sequence was identical, and secondary and tertiary structures are similar between the proposed biosimilar and the reference products. Insulin receptor binding affinity and phosphorylation studies also established analytical similarity. MYL-1501D demonstrated high similarity in different metabolic assays of glucose uptake, adipogenesis activity, and inhibition of stimulated lipolysis. Rabbit bioassay studies showed MYL-1501D and EU-approved insulin glargine are highly similar to US-licensed insulin glargine. These product quality studies show high similarity between MYL-1501D and licensed or approved insulin glargine products and suggest the potential of MYL-1501D as an alternative cost-effective treatment option for patients and clinicians.

Buried Kex2 Sites in Glargine Precursor Aggregates Prevent Its Intracellular Processing in Pichia pastoris Muts Strains and the Effect of Methanol-Feeding Strategy and Induction Temperature on Glargine Precursor Production Parameters.

Glargine is a long-acting insulin analog with less hypoglycemia risk. Like human insulin, glargine is a globular protein composed of two polypeptide chains linked by two disulfide bonds. Pichia pastoris KM71 Muts strains were engineered to produce and secrete insulin glargine through the cleavage of two Kex2 sites. Nevertheless, the recombinant product was the single-chain insulin glargine (glargine precursor) instead of the expected double-chain glargine. Molecular model analysis of the dimeric and hexameric forms of the single-chain glargine showed buried Kex2 sites that prevent intracellular glargine precursor processing. The effect of the methanol-feeding strategy (methanol limited fed-batch vs. methanol non-limited fed-batch) and the induction temperature (28 °C vs. 24 °C) on the cell growth and production parameters in bioreactor cultures was also evaluated. Exponential growth at a constant specific growth rate was observed in all the cultures. The volumetric productivities and specific substrate consumption rates were directly proportional to the specific growth rate. The lower temperature led to increased metabolic activity of the yeast cells, which increased the specific growth rate. The methanol non-limited fed-batch culture at 24 °C showed the highest values for the process parameters. After 75 h of induction, 0.122 g/L of glargine precursor was obtained from the culture medium.

Determination of amyloid core regions of insulin analogues fibrils.

A rapid-acting insulin lispro and long-acting insulin glargine are commonly used for the treatment of diabetes. Clinical cases have described the formation of injectable amyloidosis with these insulin analogues, but their amyloid core regions of fibrils were unknown. To reveal these regions, we have analysed the hydrolyzates of insulin fibrils and its analogues using high-performance liquid chromatography and mass spectrometry methods and found that insulin and its analogues have almost identical amyloid core regions that intersect with the predicted amyloidogenic regions. The obtained results can be used to create new insulin analogues with a low ability to form fibrils. ABBREVIATIONS: a.a., amino acid residues; HPLC-MS, high-performance liquid chromatography/mass spectrometry; m/z, mass-to-charge ratio; TEM, transmission electron microscopy.

Concentration and Chemical Stability of Commercially Available Insulins: A High-Resolution Mass Spectrometry Study.

Adequacy of insulin concentration in commercially available insulin formulations has recently been challenged. We therefore repeatedly evaluated insulin content and stability of 58 insulin vials containing 5 different insulin formulations (human insulin, standard/faster-acting insulin aspart, insulin lispro, and insulin glargine) over a period of 85 days. High-resolution mass spectrometry was used to quantify intact monomeric insulin in glass vials and plastic pump cartridges exposed to three different temperatures (4°C, 22°C, 37°C), simulating real-life conditions. In all cases, measured insulin concentration was in accordance with FDA and European Medicines Agency (EMA) requirements without evidence of chemical instability.

Update on Biosimilar Insulins: A US Perspective.

The development of biosimilar insulin products has slowly evolved with only two follow-on biologics currently available to patients in the US. Both Basaglar® (insulin glargine) and Admelog® (insulin lispro) have undergone extensive testing, and have gained significant use by patients in the US. Despite the availability of these follow-on products, the price of insulin has remained stubbornly high. New regulatory guidance under the Biologics Price Competition and Innovations Act that came into effect in March 2020 introduced an abbreviated pathway for the approval of biosimilar insulins and introduced the option to apply for interchangeability of the biosimilar insulin with the reference product. This abbreviated clinical testing may open the doors for numerous follow-on insulin products, with unknown supply-chain and fiscal ramifications. This review will highlight the development process of biosimilar insulin in the US and the recent regulatory changes that can aid this process. We will also discuss challenges for prescribers and patients who are navigating this ever-changing landscape. These new regulations for biosimilar insulins will have ramifications for patients, healthcare providers, and third-party payers, though the direction and scope of these changes is unclear.

The quaternary structure of insulin glargine and glulisine under formulation conditions.

The quaternary structures of insulin glargine and glulisine under formulation conditions and upon dilution using placebo or water were investigated using synchrotron small-angle X-ray scattering. Our results revealed that insulin glulisine in Apidra® is predominantly hexameric in solution with significant fractions of dodecamers and monomers. Upon dilution with placebo, this equilibrium shifts towards monomers. Insulin glargine in Lantus® and Toujeo® is present in a stable hexamer/dimer equilibrium, which is hardly affected by dilution with water down to 1 mg/ml insulin concentration. The results provide exclusive insight into the quaternary structure and thus the association/dissociation properties of the two insulin analogues in marketed formulations.

Identification of recombinant human insulin and biosynthetic insulin analogues by multiplexed targeted unlabeled mass spectrometry of proteotypic tryptic peptides.

Direct qualitative methods that allow the rapid screening and identification of insulin products during early stages of the drug development process and those already in the market can be of great utility for manufacturers and regulatory agencies and the recent scientific literature describes several methods. Herein, a qualitative proteomic method is presented for the identification of recombinant human insulin and all marketed biosynthetic analogues -insulin lispro, aspart, glulisine, glargine, detemir and degludec- via tryptic digestion and identification of proteotypic peptides for each insulin. Individual insulins were first denatured under reducing conditions and the cysteine residues blocked by iodoacetamide. The proteins were then digested with trypsin and the peptide products separated by reversed phase liquid chromatography on an Ascentis® Express ES-C18 column and detected by positive polarity ESI-MS/MS. The digestion peptides were characterized using a multiplexed MRM approach that monitors the fragmentation of the doubly charged unlabeled precursor ion of each peptide into a collection of signature y and b ions. The MRM transitions for the individual peptides were optimized to allow maximal ionization on a standard triple quadrupole mass spectrometer. All products of the digestion procedure for all insulins were detected with adequate signal intensity except for the C-terminal B30Thr whenever it was present and cleaved and the tryptic B1-3 tripeptide of insulin glulisine. The unique proteotypic peptides identified for each of the insulin analogues coupled with their signature y and b ions permitted the unambiguous verification of all sequence variations and chemical modifications. The elution of the A polypeptide chain for all insulins and the tryptic peptides of the B chain, with the exception of a very few, occurred around the same time point. This underscores the close similarity in the physicochemical properties between the digestion peptides and is consistent with the subtle variations in amino acid sequence among the various insulins. Therefore, the identification and distinction of the different types of insulin based solely on the chromatographic retention time of their respective proteolytic products can be deceptive without proper mass spectrometric analysis and may result in false positives.

Evaluation of the innate immunostimulatory potential of originator and non-originator copies of insulin glargine in an in vitro human immune model.

BACKGROUND: The manufacture of insulin analogs requires sophisticated production procedures which can lead to differences in the structure, purity, and/or other physiochemical properties of resultant products that can affect their biologic activity. Here, we sought to compare originator and non-originator copies of insulin glargine for innate immune activity and mechanisms leading to differences in these response profiles in an in vitro model of human immunity. METHODS: An endothelial/dendritic cell-based innate immune model was used to study antigen-presenting cell activation, cytokine secretion, and insulin receptor signalling pathways induced by originator and non-originator insulin glargine products. Mechanistic studies included signalling pathway blockade with specific inhibitors, analysis of the products in a Toll-like receptor reporter cell line assay, and natural insulin removal from the products by immunopurification. FINDINGS: All insulin glargine products elicited at least a minor innate immune response comparable to natural human insulin, but some lots of a non-originator copy product induced the elevated secretion of the cytokines, IL-8 and IL-6. In studies aimed at addressing the mechanisms leading to differential cytokine production by these products, we found (1) the inflammatory response was not mediated by bacterial contaminants, (2) the innate response was driven by the native insulin receptor through the MAPK pathway, and (3) the removal of insulin glargine significantly reduced their capacity to induce innate activity. No evidence of product aggregates was detected, though the presence of some high molecular weight proteins argues for the presence of insulin glargine dimers or others contaminants in these products. CONCLUSION: The data presented here suggests some non-originator insulin glargine product lots drive heightened in vitro human innate activity and provides preliminary evidence that changes in the biochemical composition of non-originator insulin glargine products (dimers, impurities) might be responsible for their greater immunostimulatory potential.

Insulin glargine and its two active metabolites: A sensitive (16pM) and robust simultaneous hybrid assay coupling immunoaffinity purification with LC-MS/MS to support biosimilar clinical studies.

MK-1293 is a newly approved follow-on/biosimilar insulin glargine for the treatment of Type 1 and Type 2 diabetics. To support pivotal clinical studies during biosimilar evaluation, a sensitive, specific and robust liquid chromatography and tandem mass spectrometry (LC-MS/MS) assay for the simultaneous quantification of glargine and its two active metabolites, M1 and M2 were developed. Strategies to overcome analytical challenges, so as to optimize assay sensitivity and improve ruggedness, were evolved, resulting in a fully validated LC-MS/MS method with a lower limit of quantification (LLOQ) at 0.1ng/mL (∼16pM, equivalent to ∼2.8µU/mL) for glargine, M1 and M2, respectively, using 0.5mL of human plasma. The assay employed hybrid methodology that combined immunoaffinity purification and reversed-phase chromatography followed by electrospray-MS/MS detection operated under positive ionization mode. Stable-isotope labeled 6[D10]Leu-glargine and 4[D10]Leu-M1 were used as internal standards. With a calibration range from 0.1 to 10ng/mL, the intra-run precision (n=5) and accuracy were <6.21%, and 96.9-102.1%, while the inter-run (n=5/run for 7days) precision and accuracy were <9.55% and 96.5-105.1%, respectively, for all 3 analytes. Matrix effect, recovery, analyte stability, and interferences from control matrix, potential concomitant medications and anti-drug antibody were assessed. The assay was fully automated and has been successfully used in support of biosimilar clinical studies. Greater than 94.3% of incurred sample reanalysis (ISR) results met acceptance criteria, demonstrating the robustness of the assay. The strategic considerations during method development and validation are discussed, and can be applied to quantification of other peptides, especially insulin analogs, in the future.

Glargine and degludec: Solution behaviour of higher dose synthetic insulins.

Single, double and triple doses of the synthetic insulins glargine and degludec currently used in patient therapy are characterised using macromolecular hydrodynamic techniques (dynamic light scattering and analytical ultracentrifugation) in an attempt to provide the basis for improved personalised insulin profiling in patients with diabetes. Using dynamic light scattering and sedimentation velocity in the analytical ultracentrifuge glargine was shown to be primarily dimeric under solvent conditions used in current formulations whereas degludec behaved as a dihexamer with evidence of further association of the hexamers ("multi-hexamerisation"). Further analysis by sedimentation equilibrium showed that degludec exhibited reversible interaction between mono- and-di-hexamer forms. Unlike glargine, degludec showed strong thermodynamic non-ideality, but this was suppressed by the addition of salt. With such large injectable doses of synthetic insulins remaining in the physiological system for extended periods of time, in some case 24-40 hours, double and triple dose insulins may impact adversely on personalised insulin profiling in patients with diabetes.

Efficacy and Safety of Biosimilar SAR342434 Insulin Lispro in Adults with Type 1 Diabetes Also Using Insulin Glargine-SORELLA 1 Study.

BACKGROUND: SAR342434 is a biosimilar follow-on of insulin lispro-Humalog®. This study aimed to show similar efficacy, safety, and immunogenicity of SAR342434 (SAR-Lis) versus insulin lispro-Humalog (Ly-Lis) in adult patients with type 1 diabetes (T1DM) treated with multiple daily injections while using basal insulin glargine (Lantus®; GLA-100). MATERIALS AND METHODS: SORELLA-1 was a randomized, open-label phase 3 study (NCT02273180). Patients completing the 6-month main study continued on SAR-Lis or Ly-Lis, as randomized, for a 6-month safety extension. Assessments included change in HbA1c, fasting plasma glucose (FPG), seven-point self-monitored plasma glucose (SMPG) profiles, hypoglycemic events, treatment-emergent adverse events (TEAEs), and anti-insulin antibodies (AIAs). RESULTS: Five hundred seven patients were randomized (SAR-Lis n = 253; Ly-Lis n = 254). Least square (LS) mean (SEM) change in glycosylated hemoglobin (HbA1c) (baseline to week 26; primary endpoint) was similar in both treatment groups (SAR-Lis: -0.42% [0.051]; Ly-Lis: -0.47% [0.050]). Noninferiority at prespecified 0.3% noninferiority margin and inverse noninferiority were demonstrated (LS mean difference of SAR-Lis vs. Ly-Lis: 0.06% [95% confidence interval: -0.084 to 0.197]). At week 52 (end of extension period) versus week 26, a small HbA1c increase was observed in both groups. FPG and seven-point SMPG profile changes, including postprandial glucose excursions, were similar between groups. At week 52, similar changes in mean daily mealtime and basal insulin doses were observed. Hypoglycemia, TEAEs, and AIAs (incidence, prevalence) did not differ between groups. CONCLUSIONS: Results from this controlled study in patients with T1DM also using GLA-100 support similar efficacy and long-term safety (including immunogenicity) of SAR-Lis and Ly-Lis.

Insulin glargine 300 units/mL: A new basal insulin product for diabetes mellitus.

PURPOSE: The pharmacokinetics, efficacy, and safety of U-300 insulin glargine for the management of diabetes are reviewed. SUMMARY: U-300 (300 units/mL) insulin glargine is a long-acting basal insulin with low within-day variability, high day-to-day reproducibility, longer duration, and constant pharmacokinetic profile compared with U-100 (100 units/mL) insulin glargine. U-300 was evaluated in six randomized, active-comparator, open-label, Phase III clinical studies (EDITION trials) among patients with type 1 or 2 diabetes. The primary endpoint for all EDITION studies was the reduction in glycosylated hemoglobin from baseline to six months. Safety endpoints included confirmed or nocturnal hypoglycemia between week 9 and month 6 and the change in weight from baseline. For hypoglycemic episodes, U-300 insulin glargine was superior to U-100 insulin glargine when comparing the risk of hypoglycemia. U-300 insulin glargine is supplied in a prefilled device (for safety purposes) and packaged in boxes of three or five pens. It is still early to determine the role of U-300 insulin glargine in diabetes management. When compared with U-100 insulin glargine, U-300 insulin glargine appeared to be associated with a lower risk of hypoglycemia and nocturnal hypoglycemia, most likely due to its pharmacokinetics. The wholesale average cost of U-300 insulin glargine is $335.48 per box of three pens. CONCLUSION: The efficacy outcomes of U-300 insulin glargine were similar to those of U-100 insulin glargine, but the constant pharmacokinetic profile and longer duration of action of U-300 insulin glargine may help certain patients with type 1 or type 2 diabetes achieve better glycemic control.

Recombinant Glargine Insulin Production Process Using Escherichia coli.

Glargine insulin is a long-acting insulin analog that helps blood glucose maintenance in patients with diabetes. We constructed the pPT-GI vector to express prepeptide glargine insulin when transformed into Escherichia coli JM109. The transformed E. coli cells were cultured by fed-batch fermentation. The final dry cell mass was 18 g/l. The prepeptide glargine insulin was 38.52% of the total protein. It was expressed as an inclusion body and then refolded to recover the biological activity. To convert the prepeptide into glargine insulin, citraconylation and trypsin cleavage were performed. Using citraconylation, the yield of enzymatic conversion for glargine insulin increased by 3.2-fold compared with that without citraconylation. After the enzyme reaction, active glargine insulin was purified by two types of chromatography (ion-exchange chromatography and reverse-phase chromatography). We obtained recombinant human glargine insulin at 98.11% purity and verified that it is equal to the standard of human glargine insulin, based on High-performance liquid chromatography analysis and Matrix-assisted laser desorption/ionization Time-of-Flight Mass Spectrometry. We thus established a production process for high-purity recombinant human glargine insulin and a method to block Arg (B31)-insulin formation. This established process for recombinant human glargine insulin may be a model process for the production of other human insulin analogs.