Calibration-free concentration analysis for quantification of anti-drug specific antibodies in polyclonal positive control antibodies and in clinical samples.

Highly sensitive assays for anti-drug antibodies (ADAs) are both a regulatory requirement and requisite for proper evaluation of the effects of immunogenicity on clinical efficacy and safety. Determination of ADA assay sensitivity depends on positive control antibodies to represent naturally occurring or treatment-induced ADA responses. An accurate determination of the proportion of drug-specific antibodies in these polyclonal positive control batches is critical for correct evaluation of assay sensitivity. Target purification of positive control antibodies is commonly applied but infers the risk to lose a proportion of the antibodies. This may lead to an incorrect estimate of the ADA assay sensitivity, especially if high-affinity antibodies are lost that may be representative of natural ADAs with clinical implication. The Surface Plasmon Resonance platform on the Biacore™ systems offers methods for real-time analysis of biomolecular interactions without introducing any modifications to the analysed material. Calibration-free concentration analysis (CFCA) is such an application for determination of the proportion of drug-specific antibodies, which allows direct determination of active antibody concentrations, as defined by the ligand, in a flow-based system. Here, we present a novel CFCA method for ADA quantification developed and validated using polyclonal positive control antibodies against endogenous human insulin, insulin degludec (Tresiba®) and turoctocog alfa (NovoEight®). We find that CFCA precisely and accurately measures concentrations of drug-specific IgG antibodies with a precision of ±10% and 90%-112% recovery of expected values of monoclonal positive control antibodies. Additionally, we have achieved a more accurate measure of the sensitivity of a cell-based bioassay for in vitro neutralising ADAs using the specific concentration determined with CFCA. Moreover, we effectively quantified serum anti-insulin antibodies in high-titre clinical samples from individuals with diabetes mellitus. This application extends the relevance of the CFCA technology to analysis of immunogenicity for accurate quantification of ADAs in both the polyclonal positive control and in clinical samples.

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.

Direct comparison of radioimmunoassay and LC-MS/MS for PK assessment of insulin glargine in clinical development.

BACKGROUND: A direct comparison of radioimmunoassay (RIA) and LC-MS/MS for insulin glargine quantification in human plasma is provided. RESULTS: Compared with the RIA, the LC-MS/MS assay exhibited comparable/improved sensitivity (LLOQ at 0.1 ng/ml [˜16.7 pM or 2.8 µU/ml] for glargine and its metabolites M1 and M2, respectively) and ruggedness. Most importantly, it demonstrated a superior specificity advantage against the interference from endogenous insulin, exogenous insulin analogs (e.g., Novolog(®), Humalog(®) or Levemir(®), routine treatment for diabetes mellitus) and potentially pre-existing anti-insulin antibodies in patient samples. The data obtained from diabetic patients suggested the LC-MS/MS assay substantially improved pharmacokinetic characterization of glargine. CONCLUSION: LC-MS/MS overcame common limitations of RIA, and provided critically needed specificity to support glargine clinical development, without sacrificing assay sensitivity and ruggedness.

Extensive study of human insulin immunoassays: promises and pitfalls for insulin analogue detection and quantification.

BACKGROUND: Over the last few decades, new synthetic insulin analogues have been developed. Their measurement is of prime importance in the investigation of hypoglycaemia, but their quantification is hampered by variable cross-reactivity with many insulin assays. For clinical analysis, it has now become essential to know the potential cross-reactivity of analogues of interest. METHODS: In this work, we performed an extensive study of insulin analogue cross-reactivity using numerous human insulin immunoassays. We investigated the cross-reactivity of five analogues (lispro, aspart, glulisine, glargine, detemir) and two glargine metabolites (M1 and M2) with 16 commercial human insulin immunoassays as a function of concentration. RESULTS: The cross-reactivity values for insulin analogues or glargine metabolites ranged from 0% to 264%. Four assays were more specific to human insulin, resulting in negligible cross-reactivity with the analogues. However, none of the 16 assays was completely free of cross-reactivity with analogues or metabolites. The results show that analogue cross-reactivity, which varies to a large degree, is far from negligible, and should not be overlooked in clinical investigations. CONCLUSIONS: This study has established the cross-reactivity of five insulin analogues and two glargine metabolites using 16 immunoassays to facilitate the choice of the immunoassay(s) and to provide sensitive and specific analyses in clinical routine or investigation.

Experimental testing of skin reactions to insulin detemir in diabetes patients naïve to insulin detemir.

BACKGROUND/AIMS: Sporadic reports on immediate and delayed cutaneous reactions to insulin detemir, a modern insulin analogue, have raised unsupported claims of allergy of type I, III and IV. The purpose of this experimental study using a provocative design was to elucidate the potential mechanisms behind such skin reactions. MATERIAL AND METHODS: A total of 40 patients with type 1 diabetes or insulin-requiring type 2 diabetes, all naïve to insulin detemir, were injected on the thigh with 0.l mL of insulin detemir (Levemir(®)) administered with an 8 mm needle at three different depths, i.e. intradermal, subdermal and subcutaneously. Saline was injected as control. Any cutaneous reactions were assessed after 10 and 30 min, after 24 and 48 h and after 7 days. Histopathology of positive reactions on day 7 was obtained. The study was randomized, controlled, double-blinded, and conducted in accordance with ICH-GCP guidelines. Blood flow was recorded with the Periflux PF5010, and skin colour (a*) with the DSMII colorimeter. RESULTS: Clinical reading, flowmetry and colorimetry consistently showed delayed reactions after intradermal insulin injection (35 of 40 patients reacted with mainly weak reactions, P<0.05), peaking after 48 h, contrasting no special reaction immediately after injection, except for reactions attributed to needle trauma. A total of 22 patients reacted on subdermal injection and 21 on subcutaneous injection. Histopathology on day 7 from 22 reactions in 15 patients showed a consistent pattern of inflammation with eosinophilia as typically observed in adverse skin reactions to a variety of medicines. Reactions were interpreted as non-specific biologic responses to the insulin different from direct toxic actions and classical allergic reaction patterns. Only one person registered itch/discomfort. A prick test vs. histamine reference excluded insulin detemir to be a pharmacological histamine releaser. Thus, provocative testing with insulin detemir produced delayed skin reaction but no immediate reaction. Measurement of circulating insulin detemir-specific antibodies by RIA before and after 3 months showed no increase. CONCLUSION: Non-allergic delayed skin reactions from intradermal and, to a minor degree, subdermal and subcutaneous injections of insulin detemir were frequent in this experimental study and showed a consistent histology pattern of inflammation with eosinophilia. Immediate reactions were not produced. The reactions are unlikely to be specific for insulin detemir, and other insulins should be studied in a similar provocative design.

Insulin allergy; desensitization with crystalline zinc-insulin and steroid tapering.

The insulin analogues, aspart and lispro, have been considered safe alternatives for patients with insulin allergy, because of their decreased immunogenicity. However, recent several reports showed that neither of them was completely free from allergic reactions. We also experienced a patient with insulin allergy not only to human regular insulin but also to both of the insulin analogues. Interestingly, the insulin analogues, which readily dissociate from polymer to monomer, induced the most severe allergic reaction among several types of human insulin reagents in the present case. Allergy to crystalline zinc-insulin, the three-dimensional structure of which results in delayed dissociation and absorption, was negative on intradermal tests. However, its large subcutaneous injection caused local allergic reaction. These results suggested that the allergic reaction might depend on the rapidity of insulin monomerization and absorption, and thus that the immunogenic residue of insulin is concealed when insulin is polymerized. Based on the intradermal tests, we speculated that the antigenic epitope might be B30-Thr in the present case. We also report here the modified method of insulin desensitization using crystalline zinc-insulin with prednisolone tapering. This might be a simple and useful treatment for insulin allergy.

Anaphylaxis to subcutaneous neutral protamine Hagedorn insulin with simultaneous sensitization to protamine and insulin.

We report an insulin-treated diabetic patient who suffered, in a 2-month period, three severe anaphylactic reactions immediately after self-administered subcutaneous injections of neutral protamine Hagedorn (NPH) human recombinant-DNA insulin. These reactions consisted of local and systemic symptoms, including dyspnea and hypotension. A simultaneous sensitization to human insulin and to protamine was demonstrated, both by skin tests and by the determination of serum specific IgE. Suspecting protamine allergy, we performed a test dose to human lente insulin with perfect tolerance. After a 1-year follow-up with lente-insulin treatment, no reactions have occurred, despite treatment interruptions. Therefore, protamine IgE-mediated allergy probably caused our patient's reactions. In conclusion, protamine sensitization should be ruled out in any patient with a history of reactions to subcutaneous protamine-containing insulins, even if insulin sensitization is present.

Protamine antibody production in diabetic subjects treated with NPH insulin.

Treatment with neutral protamine Hagedorn (NPH) insulin predisposes individuals with diabetes to anaphylactoid reactions when given bolus protamine for heparin reversal during cardiovascular procedures. To prospectively examine production of protamine antibodies, 30 patients with non-insulin dependent diabetes were followed for 12 months from initiation of therapy with porcine NPH or Lente insulin. Twenty-one subjects were randomly assigned to NPH (protamine containing) and nine controls to Lente (protamine free) insulin. Protamine specific IgG antibody was produced by 6/21 (29%) of NPH-treated subjects and 0/9 controls. Among NPH treated subjects, there was no difference between protamine antibody producers and non-producers with regard to age, race, weight, or pre-treatment glycosylated hemoglobin. Both producer and non-producer groups received similar amounts of insulin and protamine and achieved similar glycemic control. Insulin antibodies were made by 4/6 (67%) of protamine antibody producers and by 6/15 (40%) of non-producers (NS). The authors conclude that one of three new diabetics who are treated with porcine NPH insulin will make IgG protamine antibodies. These antibodies do not affect insulin requirements, glycemic control, or insulin antibody production. Because of the frequency of protamine antibody production and the risk of anaphylaxis to bolus protamine administration in NPH treated diabetics, the authors suggest that NPH insulin-treated individuals should avoid heparin reversal by protamine.

Comparison of insulin antibody levels during the first 3 years of treatment of adult diabetics with monocomponent porcine lente-insulin and single peak beef NPH insulin.

Insulin antibody production was studied in two groups of 9 adult diabetics each, who were never treated before with insulin. One group received Monocomponent porcine lente insulin (MC) and the other group single peak beef NPH insulin, for 3 years. Insulin antibodies were evaluated by antibody-bound immunoreactive insulin (Abl) and by the labelled insulin binding capacity (IBC) which presented a significant correlation best fitted by a logarithmic curve. The patients treated with MC insulin developed significant levels of insulin antibodies, however, at lower levels and appearing later in comparison to those with beef NPH. Only 3 patients did not produce significant levels of insulin antibodies. The highest titers occurred after different lengths of treatment in the two groups of patients. Abl decreased after continuation of treatment particularly in the MC series. While in the MC-treated patients there was some positive correlation between the insulin dose and the level of Abl no significant correlation was found between the diabetes control and insulin antibody titers in both groups of patients.

[ELISA detection of protamine antibodies]. / ELISA-Nachweis von Protaminantikörpern.

Protamine is frequently used as an adjuvant in insulin preparations. As alien protein protamine is immunogenic. We developed an ELISA for detection of protamine antibodies. As a strongly basic molecule protamine shows remarkable reactivity. Resulting methodical difficulties with regard to the assay are discussed. The course of antibody titres to protamine after immunization is shown in rabbits, goats, sheep and guinea pigs with positive results in all species. In humans protamine antibodies were detectable in 4% of 150 patients treated with protamine insulins.

[Factors affecting the antigenicity of exogenous insulin in diabetics]. / Izsledvane na niakoi faktori, koito povliiavat antigennostta na ekzogenniia insulin pri diabetno bolni.

The antigenic properties of insulin and the effect of additional factors on them, HL-A-phenotype including were studied under different conditions in 3 groups of diabetic patients (I--71 patients treated with insulin, II--48 diabetic patients--never treated with insulin and III--51 diabetics, treated at least 6 months with conventional insulin and later treated with monocomponent insulin "Actrapid"). In some other experiments, making use of 7 groups guinea pigs, the antigenicity of different trade insulin preparations was tested. The antibody titre was assessed by the insulin-binding serum capacity, incubated with 131I-insulin. The authors established a significant dependence of antibody titre on insulin during insulin treatment and insignificant--with the age of the patients, duration of insulin treatment and HL-A phenotype. The last correlation with HLA-phenotype B 15 was highly significant. A reduction of antibody titre was established after changing from conventional to highly-purified monocomponent insulin preparation and anew elevation of titre with the resumed treatment with non-purified insulin forms according to special ways. All insulin preparations, that the separate groups of guinea pigs were injected, led to significant increase of antibody titre in the experimental animals. The better purified of them, however, as "insulin Comb" and Bulgarian "neutral porcine insulin"--Pharmachim, are less antigenic. A treatment with highly purified preparations is recommended for the diabetic patients with HLA-phenotype B 15.

Human ultralente insulin.

The greater solubility of human insulin and its possible faster action have led to doubts about whether a sufficiently long acting formulation could be produced to provide a basal supply for diabetics. In a double blind crossover study in 18 diabetics human ultralente insulin was as effective as beef ultralente insulin in controlling basal plasma glucose concentrations (median 5.7 mmol/l (103 mg/100 ml) with human and 6.3 mmol/l (114 mg/100 ml) with beef ultralente insulin respectively). There was no significant difference between human and bovine insulin in the rise in plasma glucose concentration from 0400 to 0700 after an injection the previous morning and no difference between patients receiving an adequate or insufficient dose of human ultralente insulin. Bovine insulin antibody binding was reduced with human insulin (p less than 0.002), which suggests that human insulin is less antigenic than beef insulin. Once daily human ultralente insulin provides a suitable formulation for the basal insulin requirement of diabetics.

Simple method for development of sensitive and specific antiinsulin antisera for laboratory use.

Commercial sources provide good, though expensive antiinsulin antisera. We describe here a simple, fast and inexpensive method for the production of antiinsulin antisera. Purified pork insulin (Lente) was injected subcutaneously in oil/water/complete Freund adjuvant mixture. Three guinea pigs received 0.25 mg of insulin and three received 0.5 mg of insulin. Subsequent injections of the same dose were done 40 and 60 days later. Five animals developed antisera with titers superior to 1:10,000 40 days after the primary inoculation. Four out of five guinea pigs improved further their antibody titer after the 2nd and 3rd injection (p less than 0.0005). Good sensitivity was associated with titers superior to 1:50,000 and appeared only after the 2nd injection to improve further after the 3rd. Thus, four out of six animals developed antiinsulin antisera suitable for the radioimmunoassay (RIA). The antisera bound proinsulin on an equimolar basis with insulin while glucagon was not bound up to 100 ng/ml. The minimum detectable insulin concentration was about 12 pg (0.3 microU) at the optimum antiserum dilution. Six animals given a small dose of insulin (0.06 mg) developed antisera of a low titer and sensitivity, unsuitable for the RIA.

Circulating IgG antibody to protamine in patients treated with protamine-insulins.

Sera from patients with different types of protamine-insulin were assayed for IgG antibody to protamine. A high prevalence of circulating antibody was found in patients treated with either bovine isophane insulin (26 out of 28 patients; 26 of whom also had antibodies to insulin), or bovine protamine zinc insulin (27 out of 30 patients; all 30 had antibodies to insulin). In sera from 24 patients treated with highly purified porcine isophane insulin, protamine antibody was detected in nine; circulating insulin-antibody was detected in 12 patients, eight of whom had protamine-antibody; in the 12 patients with no detectable antibody to insulin, antibody to protamine was detected in only one (x2 = 8.7, p less than 0.01). This relationship between insulin and protamine antigenicity is of interest as it suggests that the protamine-insulin complex is itself immunogenic.

Insulin antibodies induced by bovine insulin therapy.

Insulin antibodies are detectable in the sera of most patients within 3-4 months of starting treatment with conventional bovine insulins. Various factors determine the immunogenicity of insulin preparations including the genetic background of the recipient. This causes wide variation in response. Solid phase absorption studies as well as competitive binding in fluid phase indicate that the antibodies formed are almost always specific for determinants shared by the endogenous human molecule. This has important implications for the metabolic effect of insulin antibodies in vivo as well as for mechanisms of autoantibody production in man.

Induction of hyperglycemia with insulin antibodies to B-chain determinants.

Insulin antibodies measured by a radioimmune method (ABR) are significantly better inducers of hyperglycemia than are insulin antibodies measured by an immune hemolysis method (ABH) when injected intraperitoneally into mice. The ability to induce hyperglycemia by an insulin antiserum can be predicted by the titer of ABR measured. ABR interact in vitro with determinants severely perturbed on nickel-insulin, partially perturbed on proinsulin and desasparagine-desalanine insulin, and unaffected on zinc-insulin or zinc-free monocomponent insulin. ABH, on the other hand, interact in vitro with determinants severely perturbed on proinsulin and desasparagine-desalanine insulin but stabilized on nickel-insulin and zinc-insulin. Since the connecting peptide of proinsulin is probably in apposition to the A-chain residues on the solvent surface, the more effective reaction of proinsulin with ABR than with ABH is submitted as evidence that ABR are directed toward residues on the B-chain surface of insulin. Because ABR are more effective inducers of hyperglycemia than are ABH, it is proposed that the degree of hyperglycemia induced by antibodies in vivo is a result of interactions with determinants on the B-chain surface of insulin. These results support the possibility that insulin in vivo is more accessible for interaction with antibodies directed to the B-chain of insulin. It is also possible that ABR, which are directed to B-chain determinants, are of higher affinity than is the affinity between insulin and receptors or that the active site of insulin for maintaining euglycemia includes the B-chain surface residues.