Competitive protein adsorption in isocratic anion exchange chromatography.

Protein chromatography is the dominant method of purification of biopharmaceuticals. Although all practical chromatography involves competitive absorption and separation of M. species, competitive protein absorption has remained inadequately understood. We previously introduced the measurement of equilibrium protein adsorption isotherms with all intensive variables held constant, including competitor concentration. In this work, we introduce isocratic chromatographic retention measurements of dynamic protein adsorption in the presence of a constant concentration of a competitor protein. These measurements are achieved by establishing a dynamic equilibrium with a constant concentration of competitor (insulin) in the mobile phase flowing through an ion exchange adsorbent column and following the behavior of a test protein (α-lactalbumin) injected into this environment. We observed decreased retention times for α-lactalbumin in presence of the competitor. The presence of competitor also reduces the heterogeneity of the sites available for adsorption of the test protein. This investigation provides an approach to fundamental understanding of competitive dynamics of multicomponent protein chromatography.

Synthesis and Identification of Biologically Active Mono-Labelled FITC-Insulin Conjugate.

Fluorescently labelling proteins such as insulin have wide ranging applications in a pharmaceutical research and drug delivery. Human insulin (Actrapid®) was labelled with fluorescein isothiocyanate (FITC) and the synthesised conjugate identified using reverse phase high performance liquid chromatography (RP-HPLC) on a C18 column and a gradient method with mobile phase A containing 0.1% trifluoroacetic acid (TFA) in Millipore water and mobile phase B containing 90% Acetonitrile, 10% Millipore water and 0.1% TFA. Syntheses were carried out at varying reaction times between 4 and 20 h. Mono-labelled FITC-insulin conjugate was successfully synthesised with labelling at the B1 position on the insulin chain using a molar ratio of 2:1 (FITC:insulin) at a reaction time of 18 h and confirmed by electrospray mass spectroscopy. Reactions were studied across a pH range of 7-9.8 and the quantities switch from mono-labelled to di-labelled FITC-insulin conjugates at a reaction time of 2 h (2:1 molar ratio) at pH > 8. The conjugates isolated from the studies had biological activities in comparison to native insulin of 99.5% monoB1, 78% monoA1, 51% diA1B1 and 0.06% triA1B1B29 in HUVEC cells by examining AKT phosphorylation levels. MonoB1 FITC-insulin conjugate was also compared to native insulin by examining cell surface GLUT4 in C2C12 skeletal muscle cells. No significant difference in the cellular response was observed for monoB1 produced in-house compared to native insulin. Therefore mono-labelled FITC-insulin at the B1 position showed similar biological activity as native insulin and can potentially be used for future biomedical applications.

Insulin discovery: A pivotal point in medical history.

The discovery of insulin in 1921 - due to the efforts of the Canadian research team based in Toronto - has been a landmark achievement in the history of medicine. Lives of people with diabetes were changed forever, considering that in the pre-insulin era this was a deadly condition. Insulin, right after its discovery, became the first hormone to be purified for human use, the first to be unraveled in its amino acid sequence and to be synthetized by DNA-recombinant technique, the first to be modified in its amino acid sequence to modify its duration of action. As such the discovery of insulin represents a pivotal point in medical history. Since the early days of its production, insulin has been improved in its pharmacokinetic and pharmacodynamic properties in the attempt to faithfully reproduce diurnal physiologic plasma insulin fluctuations. The evolution of insulin molecule has been paralleled by evolution in the way the hormone is administered. Once-weekly insulins will be available soon, and glucose-responsive "smart" insulins start showing their potential in early clinical studies. The first century of insulin as therapy was marked by relentless search for better formulations, a search that has not stopped yet. New technologies may have, indeed, the potential to provide further improvement of safety and efficacy of insulin therapy and, therefore, contribute to improvement of the quality of life of people with diabetes.

100 years of insulin: celebrating the past, present and future of diabetes therapy.

The year 2021 marks the centennial of Banting and Best's landmark description of the discovery of insulin. This discovery and insulin's rapid clinical deployment effectively transformed type 1 diabetes from a fatal diagnosis into a medically manageable chronic condition. In this Review, we describe key accomplishments leading to and building on this momentous occasion in medical history, including advancements in our understanding of the role of insulin in diabetes pathophysiology, the molecular characterization of insulin and the clinical use of insulin. Achievements are also viewed through the lens of patients impacted by insulin therapy and the evolution of insulin pharmacokinetics and delivery over the past 100 years. Finally, we reflect on the future of insulin therapy and diabetes treatment, as well as challenges to be addressed moving forward, so that the full potential of this transformative discovery may be realized.

Large scale purification and characterization of A21 deamidated variant-most prominent post translational modification (PTM) for insulins which is also widely observed in insulins pharmaceutical manufacturing and storage.

Biopharmaceutical development demands appropriate understanding of product related variants, which are formed due to post-translational modification and during downstream processing. These variants can lead to low yield, reduced biological activity, and suboptimal product quality. In addition, these variants may undergo immune reactions, henceforth need to be appropriately controlled to ensure consistent product quality and patient safety. Deamidation of insulin is the most common post-translational modification occurring in insulin and insulin analogues. AsnA21 desamido variant is also the most prominent product variant formed during human insulin manufacturing process and/or during the storage. Often, this deamidated variant is used as an impurity standard during in-process and final product analysis in the QC system. However, purification of large quantity of purified deamidated material is always being challenging due to highly similar mass, ionic, hydrophobic properties, and high structural similarity of the variant compared to the parent product. Present work demonstrates the simplified and efficient scalable process for generation of AsnA21 deamidated variant in powder form with ~96% purity. The mixed-mode property of anion exchange resin PolyQuat was utilized to purify the deamidated impurity with high recovery. Subsequent reversed-phase high performance liquid chromatography (RP-HPLC) step was introduced for concentration of product in bind elute mode. Elution pool undergone isoelectric precipitation and lyophilisation. The lyophilized product allows users for convenient use of the deamidated impurity for intended purposes. Detailed characterization by Mass spectrometry revealed deamidation is at AsnA21 and further confirmed that, structural and functional characterization as well as the biological activity of isolated variant is equivalent to insulin.

Detection of insulins in postmortem tissues: an optimized workflow based on immunopurification and LC-MS/HRMS detection.

Diabetes is a worldwide disease in perpetual expansion. Type 1 and sometimes type 2 diabetic patients require daily human insulin (HI) or analog administration. Easy access to insulins for insulin-treated diabetics, their relatives, and medical professionals can enable abuse for suicidal or homicidal purpose. However, demonstrating insulin overdose in postmortem blood is challenging. Tissue analyses are contributive, as insulins can accumulate before death or undergo only limited degradation. The present study describes an assay for HI and synthetic analogs (lispro, aspart, glulisine, detemir and degludec, glargine and its main metabolite (M1)) in liver, kidney, muscle, and injection site samples. It is based on a 5-step sample preparation (reduction of tissue sample size, homogenization, extraction, concentration, and immunopurification) associated with liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/HRMS). Selectivity and limit of detection (LOD) for all target analogs were assessed in the above matrices. LOD was determined at 25 ng/g for HI and for analogs except detemir and degludec, where LOD was 50 ng/g in kidney and injection site samples and 80 ng/g in the liver and muscle. The method was applied to13 forensic cases in which insulin use was suspected.

Simultaneous separation of insulin and six therapeutic analogues on a mixed mode column: HPLC-UV method development and application.

Human insulin and six most used therapeutic analogues are very similar in terms of retention on a reversed-phase column. Thus, the LC methods prescribed in the European Pharmacopoeia monographs for insulin and insulin analogues include many similar separation methods, which tend to be time consuming when separating individual products of insulins or are inadequate when handling a mixture. In this study, we present a simple, robust, versatile and accessible HPLC-UV separation method for identification and quantification of human insulin and its analogues in one run. The simultaneous separation and detection is possible by fine-tuning the mobile phase properties that affect the separation mechanism on a mixed mode column combining anion exchange and reversed-phase characteristics. Also developed was a simple and effective SPE sample cleaning procedure with insulin recoveries ranging from 80 to 100% for all analogues. On the other hand, the concentration of major excipients such as phenol and m-cresol fall below 1%. The two developed and validated separation methods differ in their compatibility with the use of a quaternary or binary pump, thus enabling sample characterisation independent of the HPLC solvent delivery system. The methods are compatible with the use of a mass spectrometric detector for an indisputable identification.

100th anniversary of the discovery of insulin perspective: insulin and adipose tissue fatty acid metabolism.

Insulin inhibits systemic nonesterified fatty acid (NEFA) flux to a greater degree than glucose or any other metabolite. This remarkable effect is mainly due to insulin-mediated inhibition of intracellular triglyceride (TG) lipolysis in adipose tissues and is essential to prevent diabetic ketoacidosis, but also to limit the potential lipotoxic effects of NEFA in lean tissues that contribute to the development of diabetes complications. Insulin also regulates adipose tissue fatty acid esterification, glycerol and TG synthesis, lipogenesis, and possibly oxidation, contributing to the trapping of dietary fatty acids in the postprandial state. Excess NEFA flux at a given insulin level has been used to define in vivo adipose tissue insulin resistance. Adipose tissue insulin resistance defined in this fashion has been associated with several dysmetabolic features and complications of diabetes, but the mechanistic significance of this concept is not fully understood. This review focusses on the in vivo regulation of adipose tissue fatty acid metabolism by insulin and the mechanistic significance of the current definition of adipose tissue insulin resistance. One hundred years after the discovery of insulin and despite decades of investigations, much is still to be understood about the multifaceted in vivo actions of this hormone on adipose tissue fatty acid metabolism.

The discovery of insulin in Toronto: beginning a 100 year journey of research and clinical achievement.

There has been a great deal of controversy regarding priority of discovery of insulin. Indeed, many scientists made important and, in some cases, seminal contributions to identifying the endocrine role of the pancreas and the potential for pancreatic extracts to have a glucose-lowering effect. The purpose of this article is to describe the early experiences with respect to research leading to the discovery of insulin in Toronto (ON, Canada). The experiments conducted at the University of Toronto resulted in the first demonstration that a pancreatic extract could be prepared that would consistently lower glucose, reverse ketosis and arrest the catabolic effects of type 1 diabetes. The remarkably rapid commercial production of insulin soon followed. The Toronto story begins on 17 May 1921, when Frederick Banting and Charles Best began their summer research project in the laboratory of John James Rickard Macleod, and we are now celebrating the 100th anniversary of this landmark achievement. The article herein outlines the steps leading up to the discovery of insulin and provides an overview of some of the key developments in insulin therapy over the past 100 years.

High-Throughput Screening for Insulin Secretion Modulators.

The application of forward chemical genetics to insulin secretion in high-throughput has been uncommon because of high costs and technical challenges. However, with the advancement of secreted luciferase tools, it has become feasible for small laboratories to screen large numbers of compounds for effects on insulin secretion. The purpose of this chapter is to outline the methods involved in high-throughput screening for small molecules that chronically impact pancreatic beta cell function. Attention is given to specific points in the protocol that help to improve the dynamic range and reduce variability in the assay. Using this approach in 384-well format, at least 48 and as many as 144 plates can theoretically be processed per week. This protocol serves as a guideline and can be modified as required for alternate stimulation paradigms and improved upon as new technologies become available.

Sub/supercritical fluid chromatography employing water-rich modifier enables the purification of biosynthesized human insulin.

There is a paucity of knowledge surrounding the SFC purification of human insulin. The current conventional method of insulin purification involves traditional RP-HPLC that utilises copious amounts of toxic solvents. In this study, we envisaged the development of an environmentally friendly SFC method for biosynthesized human insulin purification. Various commercially available SFC columns derived with silica, 2'ethyl pyridine, diol-HILIC, and the PFP functionalities were evaluated to determine the optimal stationary phase for purification. The PFP column gave the best results with respect to efficiencies of this important biologic that yielded average recoveries of 84%. LC-MS was used to initially detect and quantify the SFC purified standard sample of insulin (purchased) as well as the biosynthesized version. Protein sequencing was employed to verify the amino acid sequencing of the insulins; as such, the standard had a 90% probability to human insulin from the database, whereas the biosynthesized version had a 96% probability. The biological activities of both versions of the SFC purified proteins were assessed in vitro using a MTT assay. The results indicated that the biological activities of both samples were retained subsequent to SFC purification. This study successfully proposes a greener and more efficient method for the purification of insulin derivatives.

Anti-inflammatory and anti-proliferative action of adiponectin mediated by insulin signaling cascade in human vascular smooth muscle cells.

After confirmation of the presence of adiponectin (ADPN) receptors and intra-cellular binding proteins in coronary artery smooth muscle cells (VSMC), we tested the hypotheses that, in acute insulin resistance: (i) the activation/inactivation of metabolic and mitogenic insulin signaling pathways are inversely affected by ADPN and, (ii) changes in VSMC migration/proliferation rates correlate with signal activity/inactivity. In primary cultures of VSMC exposed to high glucose and palmitate plus insulin, the expression of PI-3 kinase (Akt and m-TOR), MAP-Kinase (Erk and p-38) molecules, and inflammatory markers (TLR-4 and IkB-α) were assessed with Western blot, in the absence/presence of AdipoRon (AR). Migration and proliferation rates were measured in similar experimental conditions. There were decreases of ~ 25% (p-Akt) and 40-60% (p-mTOR) expressions with high glucose/palmitate, which reversed when AR was added were. Elevations in p-Erk and p-p38 expressions were obliterated by AR. Although, no changes were detected with high glucose and palmitate, when AR was added, a decline in inflammatory activity was substantiated by a ~ 50% decrease in TLR-4 and 40-60% increase in IkBα expression. Functional assays showed 10-20% rise in VSMC proliferation with high glucose and palmitate, but addition of AR lead to 15-25% decline. The degree of VSMC migration was reduced with AR addition by ~ 15%, ~ 35% and 55%, in VSMC exposed to 5 mM, 25 mM glucose and 25 mM + 200 µM palmitate, respectively. Changes in intracellular molecular messaging in experiments mimicking acute insulin resistance suggest that anti-inflammatory and anti-atherogenic actions of ADPN in VSMC are mediated via insulin signaling pathways.

Interactions of insulin with tragacanthic acid biopolymer: Experimental and computational study.

Alternative methods for insulin delivery instead of subcutaneous injection in diabetic patients is of great essential, and biocompatible polymers are one of the most efficient vehicles for this purpose. This research aims to investigate the capability of tragacanthic acid (TA) to bind insulin and release it under physiological conditions without alteration in the structure and conformation of insulin. Interactions between TA and insulin were studied using spectroscopic techniques and computational modeling by docking and molecular dynamics simulations. Our results demonstrate an entropy-driven spontaneous interaction between insulin and TA, where hydrogen bonds act as the main enthalpic contribution. According to our findings, the weak interaction between insulin and TA provides the basis for efficient capture and appropriate release of insulin by TA as a potential part of the insulin delivery system. In conclusion, tragacanth acid can be a proper candidate for insulin delivery.

Flexible and highly ordered nanopillar electrochemical sensor for sensitive insulin evaluation.

In line with growing interest in obesity management, there has been an increase in the amount of research focused on highly sensitive analysis systems for a small number of biomarers. In this paper, we introduce the highly ordered nanopillar electrode, featuring a high aspect ratio surface area that enables enhanced electron transfer. For fabrication of the flexible electrode, gold was evaporated by electronic beam lithography on polyurethane (PU), which has high flexibility. The fabricated nanopillar is 500 nm in diameter and 1500 nm in height. Based on the highly ordered nanostructure electrode, insulin was selected as a biomarker to monitor the insulin resistance associated with obesity. To effectively analyze the insulin, the self-assembled monolayer chemical was used to introduce the enzyme catalysis-based electrochemical immunoassay, leading to the analysis of the insulin concentration range from 0.1 to 1.0 ng/mL in the real sample. The square wave voltammetry principle was used to measure HRP-based electrochemical signal both electrochemically and quantitatively. Based on the nanostructural properties of significant electrochemical behavior, we successfully analyzed insulin in the plasma sample with high sensitivity (LOD of 0.1 ng/mL) and with high reproducibility (<10%). The obtained sensitivity of nanopillar electrode is approximately 10 times (1020%) greater than that of commercial electrode. The results demonstrated that the nanopillar electrode is suitable for precise and sensitive analysis of low-level biomolecules in medical and commercial fields.

Electrochemiluminescence behaviour of silver/silver orthophosphate/graphene oxide quenched by Pd@Au core-shell nanoflowers for ultrasensitive detection of insulin.

Herein, a highly efficient electrochemiluminescence resonance energy transfer (ECL-RET) immunosensor was established for ultrasensitive insulin detection. Silver/silver orthophosphate/graphene oxide composites (Ag/Ag3PO4/GO) were prepared as sensing platform for capture-antibody (Ab1) incubation. Ag3PO4 is a novel ECL donor whose emission could be remarkably enhanced by the synergetic assistance of GO with Ag NPs. Notably, GO presented excellent electrical conductivity and ultrahigh specific surface area to improve the loading capacity Ab1 and Ag3PO4, and Ag NPs with fine biocompatibility and catalytic property could immobilize Ab1 via Ag-N bond and further hasten the electron transfer to catalyze the generation of SO4•- radicals for boosting the ECL emission of donor. To establish a new ECL-RET system, Pd@Au core-shell nanoflower was prepared as a suitable ECL acceptor which could immobilize the detection-antibody (Ab2). Due to the fine spectral overlap, Pd@Au nanoflower could significantly quench the ECL emission of Ag3PO4, causing distinct decreases in ECL intensity. The proposed ECL-RET immunosensor exhibited sensitive response to insulin in a linear range of 0.0001-80 ng/mL with a low detection limit of 0.02 pg/mL (S/N = 3), it not only provides a reliable tool for insulin detection in diagnostics of diabetes, but also lights up a new avenue for designing effective ECL-RET pairs in bioanalysis.

Aptamer-gold nanoparticle doped covalent organic framework followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for selective enrichment and detection of human insulin.

In this work, we introduced an aptamer modified Au nanoparticles doped covalent organic frameworks composite (IBAs-AuNPs/COF) to improve the property of selective enrichment of insulin from serum samples. The Au nanoparticles were immobilized on imine-based COF by in-situ reduction reaction via mussel inspired polydopamine coating, and then sulfhydryl-containing aptamers were bonded to the surface of AuNPs through an Au-S linkage. Due to the excellent adsorption property of COF and specific recognition between insulin and IBAs, the IBAs-AuNPs/COF composites show selective and satisfactory extraction property to insulin in serum samples. Excellent specifity was obtained for insulin in the presence of 50-fold interfering substances including human immunoglobulin, lysozyme and biotin. The concentrations of insulin in the range of 1.0 to 50.0 µg L-1 show good linear relationship (R2 = 0.9917) with limit of detection and limit of quantitation of 0.28 µg L-1 and 0.93 µg L-1, respectively. Then, the IBAs-AuNPs/COF composites were applied to enrich insulin in serum samples followed by analysis with matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). After the recovery experiment, the developed method shows good recoveries in range of 91.6%-112.4% with low RSD value (2.4%-9.4%, n = 3) for diabetic and healthy serum samples. The developed IBAs-AuNPs/COF composites propose a new perspective for selective and efficient enrichment of biomarkers in serum samples by functionalized COF.

Model-based monitoring of industrial reversed phase chromatography to predict insulin variants.

Downstream processing in the manufacturing biopharmaceutical industry is a multistep process separating the desired product from process- and product-related impurities. However, removing product-related impurities, such as product variants, without compromising the product yield or prolonging the process time due to extensive quality control analytics, remains a major challenge. Here, we show how mechanistic model-based monitoring, based on analytical quality control data, can predict product variants by modeling their chromatographic separation during product polishing with reversed phase chromatography. The system was described by a kinetic dispersive model with a modified Langmuir isotherm. Solely quality control analytical data on product and product variant concentrations were used to calibrate the model. This model-based monitoring approach was developed for an insulin purification process. Industrial materials were used in the separation of insulin and two insulin variants, one eluting at the product peak front and one eluting at the product peak tail. The model, fitted to analytical data, used one component to simulate each protein, or two components when a peak displayed a shoulder. This monitoring approach allowed the prediction of the elution patterns of insulin and both insulin variants. The results indicate the potential of using model-based monitoring in downstream polishing at industrial scale to take pooling decisions.

In-situ photopolymerized C4-functionalized organosilicon monoliths for reversed-phase protein separation in nano-liquid chromatography.

In this study, polyhedral oligomeric silsesquioxane (POSS)-based capillary monoliths with short alkyl chain ligand in the form of butyl (C4) were synthesized via two different polymerization routes, namely UV-initiated free radical copolymerization of methacrylate-derivatized POSS (POSS-MA) with butylmethacrylate (BMA) and UV-initiated thiol-methacrylate copolymerization of POSS-MA with butanethiol (BT). An organosilicon monolith with a pore size distribution lying on both mesoporous and macroporous scales, a lower mean pore size and a higher specific surface area was obtained with UV-initiated thiol-methacrylate polymerization. Both monoliths were then comparatively evaluated for gradient separation of proteins under reversed phase conditions in nano-liquid chromatography. The chromatographic performance was defined in terms of peak-resolution and peak capacity. Four carbon (C4) functionalized-poly(POSS-MA) monolith produced by UV-initiated thiol-methacrylate polymerization exhibited better separation performance with higher peak resolutions and peak capacities. Both, the morphological characterization of monoliths and the results of gradient separation of proteins showed that thiol-methacrylate polymerization was more suitable for the synthesis of C4 functionalized organosilicon based stationary phases for reversed-phase protein separation. The monolith prepared by thiol-methacrylate polymerization was also successfully applied for impurity analysis of two important hormones, namely insulin and genotropin. A comparison with a commercial poly(styrene-co-divinylbenzene) monolith documented the good chromatographic performance of the new BT-attached poly(POSS-MA) monolith.