Comprehensive Multidimensional Liquid Chromatography-Mass Spectrometry for the Characterization of Charge Variants of a Bispecific Antibody.

Identification and further characterization of antibody charge variants is a crucial step during biopharmaceutical drug development, particularly with regard to the increasing complexity of novel antibody formats. As a standard analytical approach, manual offline fractionation of charge variants by cation-exchange chromatography followed by comprehensive analytical testing is applied. These conventional workflows are time-consuming and labor-intensive and overall reach their limits in terms of chromatographic separation of enhanced structural heterogeneities raised from new antibody formats. For these reasons, we aimed to develop an alternative online characterization strategy for charge variant characterization of a therapeutic bispecific antibody by online mD-LC-MS at middle-up (2D-LC-MS) and bottom-up (4D-LC-MS) level. Using the implemented online mD-LC-MS approach, all medium- and even low-abundant product variants previously identified by offline fraction experiments and liquid chromatography mass spectrometry could be monitored. The herein reported automated online mD-LC-MS methodology therefore represents a complementary and in part alternative approach for analytical method validation including multiattribute monitoring (MAM) strategies by mass spectrometry, offering various benefits including increased throughput and reduced sample handling and combined protein information at intact protein and peptide level.

Detailed Analytical Characterization of a Bispecific IgG1 CrossMab Antibody of the Knob-into-Hole Format Applying Various Stress Conditions Revealed Pronounced Stability.

In recent years, a variety of new antibody formats have been developed. One of these formats allows the binding of one type of antibody to two different epitopes. This can for example be achieved by introduction of the "knob-into-hole" format and a combined CrossMab approach. Due to their complexity, these bispecific antibodies are expected to result in an enhanced variety of different degradation products. Reports on the stability of these molecules are still largely lacking. To address this, a panel of stress conditions, including elevated temperature, pH, oxidizing agents, and forced glycation via glucose incubation, to identify and functionally evaluate critical quality attributes in the complementary-determining and conserved regions of a bispecific antibody was applied in this study. The exertion of various stress conditions combined with an assessment by size exclusion chromatography, ion exchange chromatography, LC-MS/MS peptide mapping, and functional evaluation by cell-based assays was adequate to identify chemical modification sites and assess the stability and integrity, as well as the functionality of a bispecific antibody. Stress conditions induced size variants and post-translational modifications, such as isomerization, deamidation, and oxidation, albeit to a modest extent. Of note, all the observed stress conditions largely maintained functionality. In summary, this study revealed the pronounced stability of IgG1 "knob-into-hole" bispecific CrossMab antibodies compared to already marketed antibody products.

25-hydroxy-Vitamin D status: limitations in comparison and clinical interpretation of serum-levels across different assay methods.

Background: Over the last decade, clinical interest to evaluate human 25-hydroxy-vitamin D (25[OH]D) serum levels has increased exponentially. In the present study, four chemiluminescence immunoassays (CLIA), one radioimmunoassy (RIA), and one high performance liquid chromatography (HPLC) method were compared and also with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in view of 25(OH)D serum level determination. Methods: For the method comparison, blood samples from 133 consecutive patients were prospectively collected. All participants gave written informed consent for their blood samples to be used in this study. They came to the Department of Nuclear Medicine of the Central Hospital Steyr (Austria) for osteodensidometric measurement as part of their preventive medical check-up. Pearson's correlation coefficients, Bland-Altman plots, and paired t-tests were calculated. Assay-specific reference ranges were considered using blood samples from persons with normal parathormone, calcium, and total-protein values (n = 97). Results: The highest correlation was between the HPLC and the LC-MS/MS method (r = 0.96). The lowest correlation was between the cobas Vitamin D3 assay (Roche) and any of the evaluated assays (r = 0.46 - 0.63). Bland-Altman plots revealed a big negative mean bias in three assays (cobas Vitamin D3 assay [Roche]: -22.8; DiaSorin LIAISON [25[OH]D total CLIA [Diasorin]: -18.4; Diasorin 25[OH]D125 I RIA [Diasorin]: -23.8 [nmol/L]) and a much smaller positive mean bias in the other assays (ClinRep complete 25[OH]D2/D3 HPLC kit [Recipe]: 2.7; ADVIA Centaur Vitamin D total assay [Siemens]: 8.2; IDS total vitamin D assay [Immunodiagnostic Systems]: 12.1 [nmol/L]) compared to the LC-MS/MS method. Meanwhile, the manufacturer has withdrawn the cobas Vitamin D3 assay from the market. Conclusions: Poor antibody specificity with cross-reactivity to other vitamin D metabolites, incomplete extraction of the 25(OH)D analyte from the vitamin D-binding protein (DBP), and confounding matrix substances such as lipids could be potential reasons for the unacceptable performance of the cobas Vitamin D3 assay (Roche) and also the significant differences in the 25(OH)D determination between various assays. Standardization and harmonization of 25(OH)D measurements are therefore urgently needed. The widespread introduction of well standardized assays in clinical laboratories is the challenge in the next years. (Clin. Lab. 2014;60:1541-1550. DOI: 10.7754/Clin.Lab.2014.131114)

Galvano- vs. metal-ceramic crowns: up to 5-year results of a randomised split-mouth study.

Full-crown restorations made by galvano forming may be considered as highly biocompatible, stable and aesthetic restorations. Therefore, they represent an alternative to conventional metal-ceramic crowns (MC), which might be associated with an allergic or toxic reaction due to metal oxides. In current literature, there are few clinical reports available, but no comparative clinical evaluation of these two systems. Thus, the purpose of this clinical observation was to compare the long-term success of galvano-formed crowns (GC) and MC and to evaluate post-operative complications. The working hypothesis was that there was no difference in clinical success between crowns based on galvano-forming procedure or conventional metal-ceramic crowns. A prospective, randomised, double-blinded clinical trial was conducted. 48 GC and 48 MC were placed in 48 periodontal healthy patients (male = 24; female = 24) in a split-mouth design. Prosthetic parameters as technical, biological and endodontic problems were recorded. Restoration survival--MC vs. GC--was compared using a non-parametric Chi-square test by McNemar at 5% level of significance. The crown restorations were re-evaluated after an observation time of 13 to 64 months (mean = 40.5; SD = 11 months). 45 GC and 45 MC (94%) were in situ without any complications. No significant differences were found between GC and MC. Surface conditions differed only in part. Fractures of the veneering material were observed in one (2%) and two (4%) for MC and GC, respectively. The presented data indicate that GC appears to be a successful alternative to conventional MC systems.