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Background Regulatory approval for a biosimilar product is provided on the basis of its comparability to an originator product. A thorough physicochemical and functional comparability exercise is a key element in demonstrating biosimilarity. Here we report the characterization of a proposed biosimilar rituximab (GP2013) and originator rituximab. Objective To compare GP2013 with originator rituximab using an extensive array of routine analytical and extended characterization methods. Methods Primary and higher order protein structures were analyzed using a variety of methods that included high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS), peptide mapping with UV and MS detection, circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, hydrogen deuterium exchange (HDX) MS, 1D 1 H nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and differential scanning calorimetry (DSC). Charge and amino acid modifications were assessed using cation exchange chromatography (CEX) and peptide mapping using reversed-phase (RP) HPLC. Boronate affinity chromatography was used to determine the relative amount of glycation. Glycans were identified and quantified after 2-aminobenzamide (2-AB) labeling and separation using normal phase HPLC with fluorescence and MS detection, respectively. Glycan site occupancy was determined using reducing capillary electrophoresis with sodium dodecyl sulfate (CE-SDS). Size heterogeneity was determined using reducing and non-reducing CE-SDS, size exclusion chromatography (SEC) and asymmetric flow field flow fractionation (AF4). Biological characterization included a series of bioassays (in vitro target binding, antibody-dependent cell-mediated cytotoxicity [ADCC], complement-dependent cytotoxicity [CDC] and apoptosis) and surface plasmon resonance (SPR) Fc receptor binding assays. Results Intact mass analysis of GP2013 and the heavy and light chains using RP HPLC–ESI–MS revealed the expected molecular mass of rituximab. The amino acid sequence was shown to be identical between GP2013 and the originator rituximab. Further sequence confirmation using RP-HPLC-UV/MS peptide mapping showed non-distinguishable chromatograms for Lys-C digested GP2013 and originator rituximab. The higher order structure of GP2013 was shown to be indistinguishable from originator rituximab using a large panel of redundant and orthogonal methods. GP2013 and originator rituximab were comparable with regard to charge variants, specific amino acid modifications and the glycan pattern. GP2013 was also shown to have similar purity, aggregate and particle levels when compared with the originator. Functionally, and by using a comprehensive set of bioassays and binding assays covering a broad range of rituximab’s functional activities, GP2013 could not be distinguished from originator rituximab. Conclusion GP2013 was shown to be physicochemically highly similar to originator rituximab at the level of primary and higher order structure, post-translational modifications and size variants. An extensive functional characterization package indicated that GP2013 has the same biological properties as originator rituximab.

Over the past couple of years, proteomics pattern analysis has emerged as an effective method for the early diagnosis of diseases such as ovarian, breast, or prostate cancer, without identification of single biomarkers. MALDI-TOF MS, for example, offers a simple approach for fast and reliable protein profiling, especially by using carrier materials with various physical and chemical properties, in combination with a MALDI matrix. This approach is referred to as material-enhanced laser desorption/ionization (MELDI). In this paper, we report the development and application of derivatized carrier materials [cellulose, silica, poly(glycidyl methacrylate/divinylbenzene) (GMA/DVB) particles, and diamond powder] for fast and direct MALDI-TOF MS protein profiling. The applicability of MELDI for rapid protein profiling was evaluated with human serum samples. These carriers, having various hydrophobicities, resulted in characteristic mass fingerprints, even if all materials were derivatized with iminodiacetic acid (IDA) to yield an immobilized metal affinity chromatography (IMAC) functionality. Our study demonstrates that analyzing complex biological samples, such as human serum, by employing different MELDI carrier materials yielded type- and size-dependent performance variation.

The presence of numerous proteomics data and their results in literature reveal the importance and influence of proteins and peptides on human cell cycle. For instance, the proteomic profiling of biological samples, such as serum, plasma or cells, and their organelles, carried out by surface-enhanced laser desorption/ionization mass spectrometry, has led to the discovery of numerous key proteins involved in many biological disease processes. However, questions still remain regarding the reproducibility, bioinformatic artifacts and cross-validations of such experimental set-ups. The authors have developed a material-based approach, termed material-enhanced laser desorption/ionization mass spectrometry (MELDI-MS), to facilitate and improve the robustness of large-scale proteomic experiments. MELDI-MS includes a fully automated protein-profiling platform, from sample preparation and analysis to data processing involving state-of-the-art methods, which can be further improved. Multiplexed protein pattern analysis, based on material morphology, physical characteristics and chemical functionalities provides a multitude of protein patterns and allows prostate cancer samples to be distinguished from non-prostate cancer samples. Furthermore, MELDI-MS enables not only the analysis of protein signatures, but also the identification of potential discriminating peaks via capillary liquid chromatography mass spectrometry. The optimized MELDI approach offers a complete proteomics platform with improved sensitivity, selectivity and short sample preparation times.

Patients with coronary heart disease (CHD) and type 2 diabetes mellitus (T2DM) have a substantially increased risk for major cardiovascular events and mortality. Increasing physical activity and improving a healthy diet may effectively reduce cardiovascular risk factors; however, the effects are often transient. In a multicenter, 1:1 randomized controlled trial including 502 patients with combined CHD and T2DM (68 ± 8 years; 84% men), we assessed the effects of a home-based telemedicine-supported lifestyle intervention (exercise training, nutritional recommendations and health literacy training) with regular individualized feedback versus usual care. The study met its primary endpoint of reduced glycated hemoglobin after 6 months in favor of the lifestyle intervention group (mean between-group difference in the complete-case analysis ( n  = 197 and n  = 193), −0.13% (95% confidence interval, −0.25 to −0.01), P  = 0.04). When individualized feedback and health literacy training were discontinued after 6 months (while other telemedicine tools were maintained), no statistically significant between-group differences were observed at 12 months. At 12 months, 31 patients (6.2%) had a major adverse cardiovascular event (lifestyle intervention, n  = 20 (8.0%); usual care, n  = 11 (4.4%); P  = 0.15), with the main reason being hospitalization for angina or revascularization (lifestyle intervention, n  = 15; usual care, n  = 8). There were five deaths (lifestyle intervention, n  = 2; usual care, n  = 3), none of which were categorized as related to the intervention. However, three events that resulted in hospitalization were categorized as potentially related to the intervention (decompensation of heart failure, vertebral disc prolapse and inguinal hernia). In conclusion, a home-based lifestyle intervention with telemedicine support showed modest effects in patients with CHD and T2DM. ClinicalTrials.gov registration: NCT03835923 . A randomized controlled trial showed that a home-based, telemedicine-supported lifestyle intervention, which consisted of exercise training, nutrition recommendations and health literacy training, reported improved glycemic control after 6 months in patients with coronary heart disease and type 2 diabetes compared to usual care.

Aims Exercise training (ET) has been consistently shown to increase peak oxygen consumption (V̇O2) in patients with heart failure with preserved ejection fraction (HFpEF); however, inter‐individual responses vary significantly. Because it is unlikely that ET‐induced improvements in peak V̇O2 are significantly mediated by an increase in peak heart rate (HR), we aimed to investigate whether baseline peak O2‐pulse (V̇O2 × HR−1, reflecting the product of stroke volume and arteriovenous oxygen difference), not baseline peak V̇O2, is inversely associated with the change in peak V̇O2 (adjusted by body weight) following ET versus guideline control (CON) in patients with HFpEF. Methods and results This was a secondary analysis of the OptimEx‐Clin (Optimizing Exercise Training in Prevention and Treatment of Diastolic Heart Failure, NCT02078947) trial, including all 158 patients with complete baseline and 3 month cardiopulmonary exercise testing measurements (106 ET, 52 CON). Change in peak V̇O2 (%) was analysed as a function of baseline peak V̇O2 and its determinants (absolute peak V̇O2, peak O2‐pulse, peak HR, weight, haemoglobin) using robust linear regression analyses. Mediating effects on change in peak V̇O2 through changes in peak O2‐pulse, peak HR and weight were analysed by a causal mediation analysis with multiple correlated mediators. Change in submaximal exercise tolerance (V̇O2 at the ventilatory threshold, VT1) was analysed as a secondary endpoint. Among 158 patients with HFpEF (66% female; mean age, 70 ± 8 years), changes in peak O2‐pulse explained approximately 72% of the difference in changes in peak V̇O2 between ET and CON [10.0% (95% CI, 4.1 to 15.9), P = 0.001]. There was a significant interaction between the groups for the influence of baseline peak O2‐pulse on change in peak V̇O2 (interaction P = 0.04). In the ET group, every 1 mL/beat higher baseline peak O2‐pulse was associated with a decreased mean change in peak V̇O2 of −1.45% (95% CI, −2.30 to −0.60, P = 0.001) compared with a mean change of −0.08% (95% CI, −1.11 to 0.96, P = 0.88) following CON. None of the other factors showed significant interactions with study groups for the change in peak V̇O2 (P > 0.05). Change in V̇O2 at VT1 was not associated with any of the investigated factors (P > 0.05). Conclusions In patients with HFpEF, the easily measurable peak O2‐pulse seems to be a good indicator of the potential for improving peak V̇O2 through exercise training. While changes in submaximal exercise tolerance were independent of baseline peak O2‐pulse, patients with high O2‐pulse may need to use additional therapies to significantly increase peak V̇O2.

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

Exercise training (ET) has been consistently shown to increase peak oxygen consumption (V̇O ) in patients with heart failure with preserved ejection fraction (HFpEF); however, inter-individual responses vary significantly. Because it is unlikely that ET-induced improvements in peak V̇O are significantly mediated by an increase in peak heart rate (HR), we aimed to investigate whether baseline peak O -pulse (V̇O  × HR , reflecting the product of stroke volume and arteriovenous oxygen difference), not baseline peak V̇O , is inversely associated with the change in peak V̇O (adjusted by body weight) following ET versus guideline control (CON) in patients with HFpEF. This was a secondary analysis of the OptimEx-Clin (Optimizing Exercise Training in Prevention and Treatment of Diastolic Heart Failure, NCT02078947) trial, including all 158 patients with complete baseline and 3 month cardiopulmonary exercise testing measurements (106 ET, 52 CON). Change in peak V̇O (%) was analysed as a function of baseline peak V̇O and its determinants (absolute peak V̇O , peak O -pulse, peak HR, weight, haemoglobin) using robust linear regression analyses. Mediating effects on change in peak V̇O through changes in peak O -pulse, peak HR and weight were analysed by a causal mediation analysis with multiple correlated mediators. Change in submaximal exercise tolerance (V̇O at the ventilatory threshold, VT1) was analysed as a secondary endpoint. Among 158 patients with HFpEF (66% female; mean age, 70 ± 8 years), changes in peak O -pulse explained approximately 72% of the difference in changes in peak V̇O between ET and CON [10.0% (95% CI, 4.1 to 15.9), P = 0.001]. There was a significant interaction between the groups for the influence of baseline peak O -pulse on change in peak V̇O (interaction P = 0.04). In the ET group, every 1 mL/beat higher baseline peak O -pulse was associated with a decreased mean change in peak V̇O of -1.45% (95% CI, -2.30 to -0.60, P = 0.001) compared with a mean change of -0.08% (95% CI, -1.11 to 0.96, P = 0.88) following CON. None of the other factors showed significant interactions with study groups for the change in peak V̇O (P > 0.05). Change in V̇O at VT1 was not associated with any of the investigated factors (P > 0.05). In patients with HFpEF, the easily measurable peak O -pulse seems to be a good indicator of the potential for improving peak V̇O through exercise training. While changes in submaximal exercise tolerance were independent of baseline peak O -pulse, patients with high O -pulse may need to use additional therapies to significantly increase peak V̇O .