Lower Platelet Count Following Rabbit Antithymocyte Globulin Induction Is Associated With Less Acute Cellular Rejection in Heart Transplant Recipients.

BACKGROUND: Unlike lymphodepletion, a decrease in platelet count following induction immunosuppressive therapy with polyclonal rabbit antithymocyte globulin (rATG) is deemed as an adverse event. However, this phenomenon may represent a particular rATG antirejection mechanism. METHODS: This retrospective single-center study included 156 patients who received a heart transplant (HTx) between 2010 and 2018. All patients received rATG induction therapy for 5 days. Absolute lymphocyte count (ALC) and platelet counts were assessed on days 0, 7, and 14 following HTx. The primary outcome of the study was the first occurrence of acute cellular rejection (ACR) defined as grade ≥ 1B within 24 months after HTx. RESULTS: Both ALC and platelet counts decreased rapidly after induction. During the 24-month follow-up period, 17% of patients had ACR. Patients with ACR had significantly higher platelet count on day 7 (145 vs 104, P < .001) and higher ALC on day 14 (162 vs 130, P = .035) than those without rejection. Patients in the highest platelet count quartile showed more ACR (50% in quartile 4 vs 0% in quartile 1, P = .006) as well as a higher cumulative total rejection score. Univariate analysis showed that ACR was associated with platelet count on day 7, recipient age, and pretransplant cytomegalovirus IgG serology. In multivariable regression analysis, platelet count on day 7 was the most accurate predictor of ACR. CONCLUSIONS: Lower platelet count after induction with rATG is associated with less ACR. This suggests platelet involvement in antirejection mechanisms of rATG and a possible rationale for targeting platelets in future immunosuppressive strategies.

LC-MS analysis combined with principal component analysis and soft independent modelling by class analogy for a better detection of changes in N-glycosylation profiles of therapeutic glycoproteins.

Therapeutic proteins are among the top selling drugs in the pharmaceutical industry. More than 60 % of the approved therapeutic proteins are glycosylated. Nowadays, it is well accepted that changes in glycosylation may affect the safety and the efficacy of the therapeutic proteins. For this reason, it is important to characterize both the protein and the glycan structures. In this study, analytical and data processing methods were developed ensuring an easier characterization of glycoprofiles. N-glycans were (i) enzymatically released using peptide-N-glycosidase F (PNGase F), (ii) reduced, and (iii) analyzed by hydrophilic interaction liquid chromatography coupled to a high-resolution mass spectrometer (HILIC-HRMS). Glycosylation changes were analyzed in human plasma immunoglobulin G samples which had previously been artificially modified by adding other glycoproteins (such as ribonuclease B and fetuin) or by digesting with enzyme (neuraminidase). Principal component analysis (PCA) and classification through soft independent modelling by class analogy (SIMCA) were used to detect minor glycosylation changes. Using HILIC-MS-PCA/SIMCA approach, it was possible to detect small changes in N-glycosylation, which had not been detected directly from the extracted-ion chromatograms, which is current technique to detect N-glycosylation changes in batch-to-batch analysis. The HILIC-MS-PCA/SIMCA approach is highly sensitive approach due to the sensitivity of MS and appropriate data processing approaches. It could help in assessing the changes in glycosylation, controlling batch-to-batch consistency, and establishing acceptance limits according to the glycosylation changes, ensuring safety and efficacy. Graphical abstract N-glycosylation characterization using LC-MS-PCA approach.

Batch-to-batch N-glycosylation study of infliximab, trastuzumab and bevacizumab, and stability study of bevacizumab.

OBJECTIVES: Infliximab, trastuzumab and bevacizumab are among the most frequently prescribed therapeutic proteins, and like most other therapeutic proteins, are glycosylated. As differences in glycosylation may significantly change the safety and efficacy of therapeutic glycoproteins, it is extremely important to control N-glycosylation consistency. In the first part of this study, the batch-to-batch consistency of the N-glycosylation of infliximab, trastuzumab and bevacizumab was analysed. In the second part, the consistency of the N-glycosylation of bevacizumab stored in polycarbonate syringes (for off-label drug use) for 3 months was examined. METHODS: N-glycans were (i) enzymatically released using peptide-N-glycosidase F, (ii) reduced, and (iii) analysed using hydrophilic interaction liquid chromatography coupled with high-resolution mass spectrometry. Mass spectrometry data were interpreted using principal component analysis combined with two-way analysis of variance and Tukey post hoc tests. The biological activity of infliximab and trastuzumab was examined using enzyme-linked immunosorbent assays. RESULTS: The results of both studies make important contributions to the field of hospital pharmacy. All batches of the studied therapeutic glycoproteins (infliximab, trastuzumab and bevacizumab) varied considerably (especially in galactosylation), while the N-glycosylation of bevacizumab remained unchanged during 3-month storage. CONCLUSIONS: Threshold values for batch-to-batch N-glycosylation variations should be established and batch-to-batch glycosylation consistency should be regularly tested. In our study, samples with significantly different N-glycosylation profiles showed no significant variations in biological activity, suggesting that the differences are probably not therapeutically significant.

Glycan characterization of biopharmaceuticals: Updates and perspectives.

Therapeutic proteins are rapidly becoming the most promising class of pharmaceuticals on the market due to their successful treatment of a vast array of serious diseases, such as cancers and immune disorders. Therapeutic proteins are produced using recombinant DNA technology. More than 60% of therapeutic proteins are posttranslationally modified following biosynthesis by the addition of N- or O-linked glycans. Glycosylation is the most common posttranslational modifications of proteins. However, it is also the most demanding and complex posttranslational modification from the analytical point of view. Moreover, research has shown that glycosylation significantly impacts stability, half-life, mechanism of action and safety of a therapeutic protein. Considering the exponential growth of biotherapeutics, this present review of the literature (2009-2015) focuses on the characterization of protein glycosylation, which has witnessed an improvement in methodology. Furthermore, it discusses current issues in the fields of production and characterization of therapeutic proteins. This review also highlights the problem of non-standard requirements for the approval of biosimilars with regard to their glycosylation and discusses recent developments and perspectives for improved glycan characterization.

[Croatian Society for Haematology and Transfusion Medicine Guidelines on the diagnosis and management of heparin induced thrombocytopenia (HIT)]. / Smjernice Hrvatskog drustva za hematologiju i transfuzijsku medicinu u dijagnosticko-terapijskom postupku za trombocitopeniju izazvanu heparinom (HIT).

Heparin induced thrombocytopenia (HIT) is a serious complication of heparin administration. In the last decade, this clinical syndrome has come into the focus of interest, primarily because of the severe thromboembolic complications that may lead to lethal outcome. In addition, great improvements have been made in the treatment with direct thrombin inhibitors and in laboratory diagnosis of HIT. As guidelines for diagnostic and management of HIT upgrade the quality of patient treatment, activities for their development have been launched in the Republic of Croatia. Based on British Committee for Standards in Haematology (BCSH) recommendations on diagnostic and treatment of HIT from 2006, activities for the introduction of new assays for anti-heparin antibodies were launched in 2008 and 2009, including algorithm of laboratory testing for HIT, sheet for clinical assessment of HIT (4T score), and education oftransfusiologists and clinicians. Upon evaluation of the results collected during one-year period, the Croatian Society of Haematology and Transfusion Medicine nominated a task force for the development of guidelines for HIT in January 2010. Following wide-ranging discussion, the guidelines were adopted in May 2011.