In-depth comparison of N-glycosylation of human plasma-derived factor VIII and different recombinant products: from structure to clinical implications.

Essentials Glycosylation heterogeneity of recombinant proteins affects pharmacokinetics and immunogenicity. N-glycomics/glycoproteomics of plasma-derived Factor VIII and 6 recombinant FVIIIs were compared. Depending on cell line, significant differences to plasma-derived FVIII were observed. Recombinant FVIIIs expressed distinct and immunologically relevant epitopes. SUMMARY: Background/Objective Human factor VIII (FVIII) is a plasma glycoprotein, defects of which result in hemophilia A. Current substitution therapy uses FVIII products purified from human plasma or from various cell lines (recombinant FVIII) with different levels of B-domain deletion. Glycosylation is a post-translational protein modification in FVIII that has a substantial influence on its physical, functional and antigenic properties. Variation in glycosylation is likely to be the reason that FVIII products differ in their pharmacokinetics, pharmacodynamics and immunogenicity. However, the literature on FVIII glycosylation is inconsistent, preventing assembly into a coherent model. Seeking to better understand the glycosylation mechanisms underlying FVIII biology, we studied the N-glycosylation of human plasma-derived (pd)FVIII and six rFVIII products expressed in CHO, BHK or HEK cell lines. Methods FVIII samples were subjected to head-to-head detailed glycomic and glycoproteomic characterization using a combination of MALDI-MS and MS/MS, GC-MS and UPLC-UV-MSE technologies. Results/Conclusion The results of our study detail the N-glycan repertoire of pdFVIII to an unprecedented level, and for the first time, provide evidence of N-glycolylneuraminic acid (NeuGc) found on pdFVIII. Although site-specific glycosylation of rFVIII proved consistent with pdFVIII regardless of the expression system, the entire N-glycan content of each sample appeared significantly different. Although the proportion of biologically important epitopes common to all samples (i.e. sialylation and high-mannose) varied between samples, some recombinant products expressed distinct and immunologically relevant epitopes, such as LacdiNAc (LDN), fucosylated LacdiNAc (FucLDN), NeuGc, LewisX/Y and Galα1,3 Gal epitopes. rFVIII expressed in HEK cells showed the greatest glycomic differences to human pdFVIII.

Comparative analysis of marketed factor VIII products: recombinant products are not alike vis-a-vis soluble protein aggregates and subvisible particles.

Essentials Aggregation is a critical quality attribute of protein therapeutics influencing immunogenicity. Aggregates and subvisible particles in 9 recombinant factor VIII (rFVIII) products were analyzed. Major differences in aggregate and particle concentrations were detected after reconstitution. rFVIII product quality determined aggregation propensity under use-relevant stress. SUMMARY: Background Recombinant protein technologies have facilitated the development of novel factor VIII (FVIII) therapeutics with improved production efficiency, potency and half-live, and a low risk of viral transmission. The increasing number of recombinant FVIII (rFVIII) products and information on their efficacy, safety and cost allow patients and healthcare professionals to adjust treatment to individual needs. Nonetheless, 20-32% of previously untreated patients with severe hemophilia A develop inhibitory antibodies to rFVIII following treatment. The root cause of the immunogenicity of rFVIII products is not well understood. Data for human interferon and human insulin products suggest that critical quality parameters such as soluble protein aggregates (SPAs) and subvisible particles (SVPs) influence the immunogenicity of protein therapeutics. Therefore, we analyzed SPA and SVP concentrations in commercially available rFVIII products and determined how these parameters change upon exposure of rFVIII products to relevant stress conditions. Objectives Compare critical quality parameters such as SPA and SVP concentrations in rFVIII products under intended use and use-relevant stress conditions. Methods Nine rFVIII products (≥ 3 lots each) were analyzed by high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) and flow cytometry-based particle analysis. Results/conclusions SPAs and SVPs were present at different concentrations in all freshly reconstituted rFVIII products: SPA concentrations ranged from 0.2% to 11.6%; SVPs were 0.7 × 106 to 114.0 × 106 / 1000 IU. Under use-relevant stress conditions (agitation and shear stress) the products formed additional SPAs and SVPs to different degrees. The collected data indicate that product quality determines its propensity to form SVPs and SPAs, and highlights differences between marketed rFVIII products.

Immune reactivity towards insulin, its amyloid and protein S100B in blood sera of Parkinson's disease patients.

Peripheral immune responses can be sensitive indicators of disease pathology. We evaluated the autoimmune reactions to endocrine (insulin) and astrocytical (S100B) biomarkers in the blood sera of 26 Parkinson's disease (PD) patients compared with controls by using ELISA. We found a statistically significant increase of the autoimmune responses to both antigens in PD patients compared with controls with a mean increase of 70% and 50% in the autoimmune reactions towards insulin and S100B, respectively. Heterogeneity of the immune responses observed in patients may reflect the modulating effect of multiple variables associated with neurodegeneration and also changes in the basic mechanisms of individual autoimmune reactivity. We did not detect any pronounced immune reactions towards insulin amyloid fibrils and oligomers in PD patients, indicating that an amyloid-specific conformational epitope is not involved in immune recognition of this amyloid type, while sequential epitope of native insulin is hidden within the amyloid structures. Immune reactions towards S100B and insulin may reflect the neurodegenerative brain damaging processes and impaired insulin homeostasis occurring in PD.

Intermediate amyloid oligomers of lysozyme: Is their cytotoxicity a particular case or general rule for amyloid?

In the current study we investigated the molecular mechanisms of cytotoxicity of amyloid oligomers of horse milk lysozyme. We have shown that lysozyme forms soluble amyloid oligomers and protofibrils during incubation at pH 2.0 and 4.5 and 57 degrees C. These structures bind the amyloid-specific dyes thioflavin T and Congo Red, and their morphology and size were analyzed by atomic force microscopy. Monomeric lysozyme and its fibrils did not affect the viability of three cell types used in our experiments including primary murine neurons and fibroblasts, as well as neuroblastoma cell line IMR-32. However, soluble amyloid oligomers of lysozyme caused death of all these cell types, as estimated by flow-cytometry counting dead cells stained with ethidium bromide. The primary cell cultures appeared to be more sensitive to amyloid than neuroblastoma cell line IMR-32. Amyloid cytotoxicity depends on the size of oligomeric particles: samples containing 20-mers formed at pH 4.5 were more toxic than tetramers and octamers present in the solution at pH 2.0. Soluble amyloid oligomers can self-assemble into doughnut-like structures; however, no correlation was observed between the amount of the doughnut-like structures in the sample and its cytotoxicity. The fact that the intermediate oligomers of such an abundant protein as lysozyme display cytotoxicity confirms a hypothesis that cytotoxicity is a common feature of protein amyloid. Inhibition of intermediate oligomer formation is crucial in preventing amyloid pathogeneses.

Amyloidogenic properties of the artificial protein albebetin and its biologically active derivatives. The role of electrostatic interactions in fibril formation.

The artificial protein albebetin (ABB) and its derivatives containing biologically active fragments of natural proteins form fibrils at physiological pH. The amyloid nature of the fibrils was confirmed by far UV circular dichroism spectra indicating for rich beta-structure, thioflavin T binding assays, and examination of the obtained polymers by atomic force microscopy. Fusing of short peptides--octapeptide of human alpha(2)-interferon (130-137) or hexapeptide HLDF-6 (41-46) of human leukemia differentiation factor--with the N-terminus of ABB led to increased amyloidogenicity of the protein: the rate of fibril formation increased and the morphology of fibrils became more complex. The presence of free hexapeptide HLDF-6 in the ABB solution had the same effect. Increasing ionic strength also activated the process of amyloid formation, but to less extent than did the peptides fused with ABB or added to the ABB solution. We suggest an important role of electrostatic interactions in formation of ABB fibrils. The foregoing ways (addition of salt or peptides) allow decrease in electrostatic repulsion between ABB molecules carrying large negative charge (-12) at neutral pH, thus promoting fibril formation.

Autoimmune responses to amyloid structures of Abeta(25-35) peptide and human lysozyme in the serum of patients with progressive Alzheimer's disease.

We have found an increased level of serum antibodies to the prefibrillar structures of both Abeta(25-35) peptide and human lysozyme in Alzheimer's disease (AD) patients compared to age-matched controls, indicating that autoimmunity is implicated in AD. In the serum of AD patients with a long-term duration (>15 years) the titer of serum antibodies to aggregates of Abeta(25-35) peptide increased by approximately 5-fold, whilst the antibody titer to lysozyme protofilaments decreased by approximately 8-fold compared to patients with AD duration of <5 years. The content of immunoglobulins of the A, G and M types declined, particularly in AD duration of >15 years. An increase in the concentration of immune complexes and higher lysozyme activity was detected in the serum of all patients and this was suggestive of an inflammatory reaction. We propose that the autoimmune response to different amyloid structures in AD can be viewed as a clearance pathway targeting amyloid development. Autoimmune response can be exploited as a marker of ongoing protein aggregation and hence be used as a diagnostic feature of AD.