Comparative study on the deamidation of three recombinant human insulins using capillary electrophoresis.

The applicability of capillary zone electrophoresis (CZE) for the separation of different recombinant human insulins and their deamidated isoforms was studied. The high resolving power of CZE is demonstrated by its ability to separate insulin isoforms differing only by 0.984 Da (different-fold deamidated forms) and even components having the exacts same mass but slightly different shapes (same-fold deamidated forms). From among the several insulins available, humulin, glargine and glulisine were selected for our study because their sequences and chemical parameters are quite similar, however, the small differences present in their amino acid sequences influence the deamidation processes. Using a background electrolyte with basic pH was favourable not only for the separation of the different types of insulin but also for the separation of deamidated protein forms even in a bare fused silica capillary. The LOD values ranged between 0.6 - 0.93 mg/L and 2.17 - 4.37 mg/L for UV and ESI-MS detection, respectively. At -20 - -80 °C, the deamidation is minimal, but at temperatures above +5 °C deamidation is accelerated. At +5 °C only 1-fold deamidation forms could be observed for each insulin. Acidified samples incubated for 1-month at room temperature showed varying levels of deamidation: 1-fold, 1-2-fold and 1-2-3-fold forms for glargine, glulisine and humulin, respectively.

Chemometrics approach for optimization of capillary electrophoretic conditions for the separation of insulin analogues.

Six insulin analogues with high degree of chemical similarity were separated using Capillary Electrophoresis (CE). In order to improve the method performance, optimization and development the Design of Experiment (DoE) approach has been used and this strategy provided related statistical models. This methodology delivered the information regarding the influence of main factors in the method development and explained the interaction of relevant factors in terms of shortening the analysis time. The response surface methodology (RSM) design employing Central Composite Face Centered (CCF) Design analyzed the effect of the most influencing factors, including background electrolyte pH and concentration, applied voltage and temperature. This study demonstrated that the approach of analyzing the influence of each parameter in the migration behavior of analyzed peptides was capable to assess the best electrophoretic conditions for the separation of insulin analogues.

Analysis of intact proteins with capillary zone electrophoresis coupled to mass spectromery using uncoated and coated capillaries.

Although, in general, the application of coated capillaries is recommended for the separation of intact proteins, bare silica capillary is still the most often used capillary due to its simplicity and cheapness. In this work, the performance of bare fused silica capillary for intact protein analysis was compared to that of different (dynamically coated polybrene (PB) and permanently coated linear polyacrylamide (LPA)) coated capillaries using capillary zone electrophoresis - mass spectrometry (CZE-MS). In cases where low pH (pH=1.8) was used in bare silica capillaries, good precision (0.56-0.78 RSD% and 1.7-6.5 RSD% for migration times and peak areas, respectively), minimal adsorption and separation efficiency (N= 27 000/m - 322 000/m) similar to or even better than those obtained with the coated capillaries (created by an intricate multi-step process) was achieved. The PB and the LPA capillaries demonstrated their slightly better resolving power in terms of separating the different forms/variants of the same protein (e.g., hemoglobin subunits). Among the studied capillaries the one with LPA coating showed the most stable separations in the long term (n=25: 0.18-0.49 RSD% and 3.1-4.9 RSD% for migration times and peak areas, respectively). For the separation of a few proteins or even a larger number of proteins in biological samples (e.g., snake venom) the application of the simple and cheap bare fused silica capillary can be considered as an efficient choice.

Determination of deamidated isoforms of human insulin using capillary electrophoresis.

The applicability of capillary zone electrophoresis (CZE) for the separation of the deamidated forms of insulin has been studied. 50 mM NH4Ac (pH=9) with 20 % v/v isopropylalcohol was found optimal for efficient separation of insulin from its even 10 deamidated forms. The developed method was efficiently applied for monitoring the degradation rate of insulin and the formation of different deamidation isoforms. Two months after the acidification more than thirty peaks can be observed in the electropherogram, because degradation products other than deamidated components were formed as well. The recorded mass spectra enabled us to assign the exact mass of the components, and thus the identification of insulin isoforms could be accomplished. We think that this study provides useful information on how the determination of several deamidation forms can be carried out with CE-MS, but the identification of the exact position of deamidation sites in the insulin molecule remains a challenge.

Application of the capillary zone electrophoresis (CZE) and capillary gel electrophoresis (CGE) for the separation of human insulin, insulin lispro and their degradation products.

Two capillary electrophoresis (CE) methods have been developed for the separation of charge and mass variants of human insulin and its recombinant analogue lispro. Since the capillary zone (CZE) and Capillary gel electrophoresis (CGE) are based on different principles of separation, they can be used to detect different impurities of insulin and its analogues. Application of CZE enabled a separation of compounds with different m/z ratio, therefore CZE is a suitable method for the separation of deamidation products of insulin. After the optimization, this method is validated according ICH requirements. CGE method was used for the separation of higher molecular weight transformation products. Experimental data have shown that CZE and CGE are simple, fast and robust methods which could be used as a routine analysis for quality control of insulin formulations.

Surgical Treatment of Hyperparathyroidism After Kidney Transplant.

Chronic renal impairment is often associated with complex bone disorders. Improvement of secondary hyperparathyroidism (HPT) is expected after kidney transplant (KT) if the glomerular filtration rate is normalized. PATIENTS AND METHODS: There were 888 KTs performed between 1996 and 2017 at our department. A total of 558 general patients have been operated on for HPT during the same period. The 2 populations had a common part: out of the 558, a total of 69 (12.4%) were in end-stage renal failure when operated on because of secondary HPT. That also means that 7.8% of all KTs were associated with HPT. Retrospective, single-center analysis was performed using the patients' medical records. The aim of our study was to analyze the results of parathyroidectomies after KT. RESULTS: Parathyroid surgery was performed on 19 patients (2.14%) because of HPT after KT. The applied surgical technique was total parathyroidectomy with autotransplant in 6 cases, subtotal parathyroidectomy in 3 cases, and selective parathyroidectomy in 10 cases. In all cases, histology revealed benign disease. Complications were observed in 10 cases (52%); there were 6 cases of postoperative hypocalcaemia (31.58%), 1 case of transient laryngeal recurrent nerve paresis (5.26%), and 6 cases of recurrent HPT (31.58%). SUMMARY: The first step of HPT management is calcimimetic drug treatment. It is essential to prevent possible complications with regular laboratory monitoring. If the proper conservative therapy is refractory or severe in complications, surgery should be chosen. If the patient is already waiting for a KT, it is worth performing the parathyroid surgery before KT. Close collaboration with endocrinologists and nephrologists is needed to achieve successful therapy.