Covalent Insulin Dimer Research Articles

Characterization of Insulin Dimers by Top-Down Mass Spectrometry.

High-molecular weight products (HMWP) are an important critical quality attribute in research and development of insulin biopharmaceuticals. We here demonstrate on two case studies of covalent insulin dimers, induced by Fe2+ incubation or ultraviolet (UV) light stress, that de novo characterization in top-down mass spectrometry (MS) workflows can identify cross-link types and sites. On the MS2 level, electron-transfer/higher-energy collision dissociation (EThcD) efficiently cleaved the interchain disulfide bonds in the dimers to reveal cross-link connectivities between chains. The combined utilization of EThcD and 213 nm ultraviolet photodissociation (UVPD) facilitated identification of the chemical composition of the cross-links. Identification of cross-link sites between chains at residue level was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds. UVPD provided identification of cross-link sites in the Fe2+-induced dimer without MS3, while cross-link site identification with MS2 was not possible for the UV light-induced dimer. Thus, using varied multistage approaches, it was discovered that in the UV light-induced dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain. In the Fe2+-induced dimer, Phe1 from both B-chains were cross-linked through a -CH2-. The UV chromophoric side chain of Phe1 was indicated in the cross-link, explaining why UVPD-MS2 was effective in fragmenting the cross-link and nearby backbone bonds. Our results demonstrated that higher-energy collisional dissociation (HCD), EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.

Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin

A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation.

Development of a spray congealing process for the preparation of insulin-loaded lipid microparticles and characterization thereof

A spray congealing process for the preparation of protein-loaded microparticles was developed. The influence of the process parameters atomization pressure and spraying temperature on particle size and process yield was investigated by experimental design. The employed spray congealing technique enabled the production of microparticles with yields ranging from 79% to 95% and median particle sizes ( d(0.5)) from 182.2 to 315 μm. Insulin lipid microparticles could be prepared without any loss of insulin during the preparation process and the protein stability was not affected by the spray congealing process as investigated by HPLC-MS analysis. The stability of insulin encapsulated in lipid microparticles under release conditions over 28 days was assessed by investigating the residual insulin content. Starting after 3 days of release, a continuous increase of desamidoinsulin in the remaining particles of up to 7.5% after 28 days was observed. An additional degradation product was detected by HPLC and HPLC-MS analysis and identified as a covalent insulin dimer by MALDI-ToF. The microparticles did not show a burst release and testing the insulin lipid microparticles in a fibrin gel chondrocyte culture revealed that the released insulin was bioactive and had a significant effect on chondrocyte extracellular matrix production.

Nanoprecipitation versus emulsion-based techniques for the encapsulation of proteins into biodegradable nanoparticles and process-related stability issues

The goal of this study was to investigate the entrapment of 3 different model proteins (tetanus toxoid, lysozyme, and insulin) into poly(D,L-lactic acid) and poly(D,L-lactic-co-glycolic acid) nanoparticles and to address process-related stability issues. For that purpose, a modified nanoprecipitation method as well as 2 emulsion-based encapsulation techniques (ie, a solid-in oil-in water (s/o/w) and a double emulsion (w(1)/o/w(2)) method) were used. The main modification of nanoprecipitation involved the use of a wide range of miscible organic solvents such as dimethylsulfoxide and ethanol instead of the common acetone and water. The results obtained showed that tetanus toxoid and lysozyme were efficiently incorporated by the double emulsion procedure when ethyl acetate was used as solvent (>80% entrapment efficiency), whereas it was necessary to use methylene chloride to achieve high insulin entrapment efficiencies. The use of the s/o/w method or the formation of a more hydrophobic protein-surfactant ion pair did not improve protein loading. The nanoprecipitation method led to a homogenous population of small nanoparticles (with size ranging from approximately 130 to 560 nm) and in some cases also improved experimental drug loadings, especially for lysozyme (entrapment efficiency > 90%). With respect to drug content determination, a simple and quick matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method provided results very close to those obtained by reverse phase-high-performance liquid chromatography. With respect to protein stability, the duration and intensity of sonication were not a concern for tetanus toxoid, which retained more than 95% of its antigenicity after treatment for 1 minute. Only a high methylene chloride:water ratio was shown to slightly decrease toxoid antigenicity. Finally, no more than 3.3% of A21 desamido insulin and only traces of covalent insulin dimer were detected in nanoparticles. In conclusion, both the double emulsion and nanoprecipitation methods allowed efficient protein encapsulation. MALDI-TOF MS allowed accurate drug content determination. The manufacturing processes evaluated did not damage the primary structure of insulin.

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry for quantitation and molecular stability assessment of insulin entrapped within PLGA nanoparticles

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) was evaluated for both qualitative and quantitative analysis of insulin entrapped within poly(D,L‐lactic‐co‐glycolic acid) nanoparticles. Quantitation was performed by adding an internal standard (arg‐insulin) to defined and unknown sample solutions, in order to reduce point‐to‐point and sample‐to‐sample variability. The ratio of the peak height of insulin to the peak height of arg‐insulin was plotted against the insulin concentration. In this way, an excellent linear relationship was found (R2 > 0.99). This method of quantitation was compared with classical UV spectroscopy and reverse‐phase high‐performance liquid chromatography measurements. All methods provided close final drug loading values for the insulin‐loaded nanoparticle batches tested. Additionally, with respect to molecular stability, covalent insulin dimers were found only at trace levels in those nanoparticles. Compared with other methods, MALDI‐TOF MS is a valuable tool for the characterization of proteins from nanoparticles, because no extensive extraction and complex sampling procedures are required. © 2005 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:688–694, 2005

Preparation and characterization of microparticles containing peptide produced by a novel process: spray freezing into liquid

The objective of this study is to evaluate excipient type on the physicochemical properties of insulin microparticles produced by spray freezing into liquid (SFL). A novel process was developed to produce microparticles containing bioactive peptides and proteins. The microparticles were formed by atomization of an aqueous feed solution containing insulin beneath the surface of a cryogenic liquid (e.g. liquid nitrogen). In this study, bovine insulin was dissolved in deionized water alone or with tyloxapol, lactose or trehalose. The aqueous solution was sprayed directly into liquid nitrogen through a polyetheretherketone capillary nozzle under high pressure to form frozen microparticles. Lyophilization was used to sublime the ice. The SFL insulin powder was characterized by different techniques, including X-ray diffraction, reverse-phase high pressure liquid chromatography, size exclusion chromatography, scanning electron microscopy (SEM), particle size distribution and surface area. The mean diameter of the insulin microparticles was 5–7 μm. SEM revealed that the microparticles were highly porous, and the morphology of the microparticles was influenced by the excipient type. The total surface area of the insulin microparticles ranged from 20 to 40 m 2/g, and the magnitude depended on the specific composition and total solids content of the aqueous feed solution. X-ray diffraction results indicated lack of crystallinity. No change in the level of the degradation product, A-21 desamido insulin, was found in the SFL insulin samples processed alone or with trehalose or tyloxapol. Similarly, no change in formation of high molecular weight transformation products (e.g. covalent insulin dimer) was detected in the samples processed with excipients. The results demonstrated that SFL is a feasible technique for forming porous microparticles containing insulin. The physicochemical properties of insulin were preserved by the SFL technique.

Porcine Insulin Biodegradable Polyester Microspheres: Stability and In Vitro Release Characteristics

The stability of porcine insulin in biodegradable polymers, i.e., poly(dl-lactide-co-glycolide) 50:50 (50:50 DL-PLGA) and poly(l-lactide) (L-PLA) was investigated. Insulin encapsulated microspheres were fabricated from both polymers using double-emulsion–solvent evaporation and emulsion–solvent evaporation techniques and subjected to accelerated stability studies at 40°C and 75% relative humidity. Porcine insulin was found to degrade in all microsphere formulations with an average of <50% of the initial loading amount remaining intact at the end of 4 weeks. The two major degradation products observed in these formulations were determined to be A-21 desamido insulin and covalent insulin dimer with trace amounts of high molecular weight transformation products. In vitro release studies in phosphate buffered saline at 37°C resulted in very slow and incomplete (<30% in 30 days) release kinetics for all microsphere formulations. Extraction and analyses of the unreleased insulin within the microspheres revealed that an average of ∼11% of the encapsulated insulin remained intact. The degradation products observed consisted of ∼15% of three distinct deamidated hydrolysis products including A-21 desamido insulin, ∼22% covalent insulin dimer, and trace amounts of high molecular weight transformation products. The degradation of porcine insulin within biodegradable polyester microspheres during stability and release studies can be attributed to the gradual decrease in the pH within the microspheres due to progressive polymer hydrolysis resulting in the production of dl-lactic and glycolic acids. The encapsulation of an acid–base indicator, bromophenol blue, in 50:50 PLGA microspheres (as a probe to estimate pH within the microspheres during accelerated stability studies) indicated that the pH decreased to ∼3.8 after 3 weeks.

Stabilization of pH-Induced Degradation of Porcine Insulin in Biodegradable Polyester Microspheres

The purpose of this research project was to stabilize the pH-induced degradation of porcine insulin encapsulated within biodegradable polyester microspheres through the incorporation of a basic additive. Insulin microspheres fabricated using Poly(L-lactide) (L-PLA) and Poly(DL-lactide-co-glycolide) (50:50 DL-PLGA) were subjected to in vitro release studies and the stability of unreleased insulin encapsulated within microspheres was investigated. The intramicrosphere pH was estimated by encapsulating acid-base indicators covering a wide pH transition range within 50:50 DL-PLGA microspheres. Finally, a basic excipient sodium bicarbonate was incorporated in 50:50 DL-PLGA microspheres to minimize acid-induced insulin degradation. The in vitro release was slow and incomplete (<30% in 30 days). Extraction and analyses of the unreleased insulin within the microspheres revealed that an average of ∼11% remained intact. The degradation products observed consisted of ∼15% of three distinct deamidated hydrolysis products including A-21 Desamido insulin, ∼22% Covalent Insulin Dimer and trace amounts of High Molecular Weight Transformation Products. Comparison of the degradation profile of unreleased insulin contained in various microsphere formulations with the in vitro release kinetics indicated that an increase in covalent dimer formation within the microspheres prior to release is associated with a decrease in the cumulative percent insulin released during a 30-day incubation period. In an attempt to correlate insulin degradation with the drop in intra-microsphere pH due to polymer hydrolysis, it was determined that the pH within a degrading microsphere reaches a value of ∼1.8 after 4 weeks. The incorporation of a basic excipient, sodium bicarbonate, in 50:50 DL-PLGA microspheres resulted in an improved in vitro release profile (cumulative release ∼47.3% in 30 days) as well as a significant reduction in covalent dimerization of the unreleased insulin to barely detectable levels. The low pH microenvironment within a degrading microsphere is one of the major factors leading to protein instability, and the degradation of proteins encapsulated within polyester microspheres can be minimized by the incorporation of a basic excipient.

Demonstration of a relatively hepatoselective effect of covalent insulin dimers on glucose metabolism in dogs

Insulin analogues with relatively greater effect on hepatic glucose production than peripheral glucose disposal could offer a more physiological approach to the treatment of diabetes mellitus. The fact that proinsulin exhibits this property to a minor degree may suggest that analogues with increased molecular size may be less able than insulin to obtain access to peripheral receptor sites. Covalent insulin dimers have previously been shown to possess lower hypoglycaemic potencies than predicted by their in vivo receptor binding affinities. Reduced rates of diffusion to peripheral target tissues might be an explanation for the lower in vivo potency compared to insulin. To test the relative hepatic and peripheral effects of covalent insulin dimers, glucose clamp procedures with D-[3-3H]glucose tracer infusions were used in anaesthetised greyhounds to establish dose-response curves for rates of hepatic glucose production and glucose disposal with insulin, N alpha B1, N alpha B'1,-suberoyl-insulin dimer, and N epsilon B29, N epsilon B'29,-suberoyl-insulin dimer. With N alpha B1, N alpha B'1,-suberoyl-insulin dimer molar potencies relative to insulin were 68%, (34-133) (mean and 95% fiducial limits), for inhibition of hepatic glucose production and 14.7%, (10.3-20.9) for glucose disposal. With N epsilon B29,N epsilon B'29,-suberoyl-insulin dimer potencies were 75%, (31-184) and 2.5%. (1.5-4.3), for inhibition of hepatic glucose production and for glucose disposal, respectively. The demonstration that both dimers exhibit a significantly greater effect on glucose production than on glucose disposal supports the suggestion that analogues with increased molecular size may exhibit reduced ability to gain access to peripheral target cells.

Demonstration of a relatively hepatoselective effect of covalent insulin dimers on glucose metabolism in dogs

Insulin analogues with relatively greater effect on hepatic glucose production than peripheral glucose disposal could offer a more physiological approach to the treatment of diabetes mellitus. The fact that proinsulin exhibits this property to a minor degree may suggest that analogues with increased molecular size may be less able than insulin to obtain access to peripheral receptor sites. Covalent insulin dimers have previously been shown to possess lower hypoglycaemic potencies than predicted by their in vivo receptor binding affinities. Reduced rates of diffusion to peripheral target tissues-might be an explanation for the lower in vivo potency compared to insulin. To test the relative hepatic and peripheral effects of covalent insulin dimers, glucose clamp procedures with D-[3-3H] glucose tracer infusions were used in anaesthetised greyhounds to establish dose-response curves for rates of hepatic glucose production and glucose disposal with insulin, NαB1, NαB′ 1,-suberoyl-insulin dimer, and NεB29, NεB′ 29,-suberoyl-insulin dimer. With NαB1, NαB′ 1,-suberoyl-insulin dimer molar potencies relative to insulin were 68%, (34–133) (mean and 95% fiducial limits), for inhibition of hepatic glucose production and 14.7%, (10.3–20.9) for glucose disposal. With NεB29,NεB′ 29,-suberoyl-insulin dimer potencies were 75%, (31–184) and 2.5%, (1.5–4.3), for inhibition of hepatic glucose production and for glucose disposal, respectively. The demonstration that both dimers exhibit a significantly greater effect on glucose production than on glucose disposal supports the suggestion that analogues with increased molecular size may exhibit reduced ability to gain access to peripheral target cells.

Chemical stability of insulin. 2. Formation of higher molecular weight transformation products during storage of pharmaceutical preparations.

Formation of covalent, higher molecular weight transformation (HMWT) products during storage of insulin preparations at 4-45 degrees C was studied by size exclusion chromatography. The main products are covalent insulin dimers (CID), but in protamine-containing preparations the concurrent formation of covalent insulin-protamine (CIP) products takes place. At temperatures greater than or equal to 25 degrees C parallel or consecutive formation of covalent oligo- and polymers can also be observed. Rate of HMWT is only slightly influenced by species of insulin but varies with composition and formulation, and for isophane (NPH) preparations, also with the strength of preparation. Temperature has a pronounced effect on CID, CIP, and, especially, covalent oligo- and polymer formation. The CIDs are apparently formed between molecules within the hexameric unit common for all types of preparations and rate of formation is generally faster in glycerol-containing preparations. Compared with insulin hydrolysis reactions (see the preceding paper), HMWT is one order of magnitude slower, except for NPH preparations.

Quantitative dissociation of glucose transport stimulation and insulin receptor tyrosine kinase activation in isolated adipocytes with a covalent insulin dimer (B29,B29′-sunberoyl-insulin)

The covalent insulin dimer B29,B29′-suberoyl-insulin was investigated for its effects on insulin receptor binding, insulin receptor tyrosine kinase activity and glucose transport in isolated adipose cells. The dimer stimulated glucose transport (initial 3- O-methylglucose uptake rate) to the same extent as insulin did (basal rate, 35 ± 3 pmol/sec/ μl lipid; insulin, 380 ± 27; B29,B29′-suberoyl-insulin, 369 ± 24, means ± S.E.), although at higher concentrations ( ec 50 1.94 ± 0.64nM versus 0.1 ± 0.02 with insulin). In contrast, the dimer only partially (23%) mimicked insulin's effect on phosphate incorporation into insulin receptors immunoprecipitated after equilibration of cells with [ 32P]phosphate. Similarly, insulin receptor tyrosine kinase as assessed by receptor autophosphorylation and phosphorylation of the substrate poly-(Glu/Tyr) was not fully activated by treatment of cells with the insulin dimer (31 and 42% of the effect of insulin, respectively) in concentrations which maximally activate glucose transport and give rise to full insulin receptor occupancy (5 × 10 −7M). Further, the dimer activated the receptor tyrosine kinase in solubilized purified insulin receptor preparations from adipose cells to only 25% of the effect of insulin ( ec 50) 32.0 ± 16 versus 1.9 ± 1.0 nM with insulin) in spite of full receptor occupancy. Binding of the dimer to insulin receptors followed single site binding kinetics, indicating that the derivative is unable to induce negative cooperativity of the insulin receptor. It is concluded that a partial phosphorylation of insulin receptors and a submaximal tyrosine kinase activation are sufficient for full stimulation of glucose transport in the adipocyte. Further, it is suggested that negative cooperativity of the insulin receptor and activation of its tyrosine kinase require a similar conformational change of the receptor protein.