In vitro field study and worldwide survey assessing how clinical haemostasis laboratories analyse recombinant and plasma-derived von Willebrand factor products.

INTRODUCTION: Several well-established clinical laboratory methods are available to measure von Willebrand factor (VWF) in plasma samples, but few data are available on their use for analysing recombinant VWF (rVWF). AIM: To evaluate how clinical diagnostic laboratories analyse rVWF and plasma-derived VWF (pdVWF) spiked in vitro into VWF-deficient plasma using quantitative protein and functional assays of VWF. METHODS: Human VWF-deficient plasma samples were spiked with rVWF (vonicog alfa; Takeda) or pdVWF/factor VIII (pdVWF/FVIII; antihemophilic factor/VWF complex [human], CSL Behring), each at final concentrations of 1.0, 0.6, 0.2, 0.1 IU/mL VWF:ristocetin cofactor activity (VWF:RCo) according to labelled VWF activity. The ISTH SSC secondary coagulation standard was used as a control. Participating laboratories received three sets of these blinded aliquots. Mean results per assay were compared with the expected potency based on the labelled VWF:RCo activity. RESULTS: Among 39 laboratories, the most commonly established assay was VWF:RCo; 22 laboratories reported data from 2214 tests. Despite a trend to lower values, VWF:RCo activities for rVWF were in agreement with target concentrations (71%-109%), whereas VWF:platelet glycoprotein Ib (VWF:GpIb) and VWF collagen-binding activity (VWF:CB) assays gave high recoveries (up to 132% and 127%, respectively). In contrast, pdVWF/FVIII was substantially underestimated by VWF:GpIb and VWF:CB assays (56%-86% recoveries), whereas the VWF:RCo assay gave recoveries of 47%-112% for pdVWF/FVIII. CONCLUSION: The results of VWF assays used in clinical laboratories differ between rVWF and pdVWF, particularly for VWF:GpIb and VWF:CB assays. These differences may arise from the higher multimeric structure of rVWF compared to pdVWF.

Macrophage Galactose Lectin Contributes to the Regulation of FVIII (Factor VIII) Clearance in Mice-Brief Report.

BACKGROUND: Although most plasma FVIII (Factor VIII) circulates in complex with VWF (von Willebrand factor), a minority (3%-5%) circulates as free-FVIII, which is rapidly cleared. Consequently, 20% of total FVIII may be cleared as free-FVIII. Critically, the mechanisms of free-FVIII clearance remain poorly understood. However, recent studies have implicated the MGL (macrophage galactose lectin) in modulating VWF clearance. METHODS: Since VWF and FVIII share similar glycosylation, we investigated the role of MGL in FVIII clearance. FVIII binding to MGL was assessed in immunosorbent and cell-based assays. In vivo, FVIII clearance was assessed in MGL1-/- and VWF-/-/FVIII-/- mice. RESULTS: In vitro-binding studies identified MGL as a novel macrophage receptor that binds free-FVIII in a glycan-dependent manner. MGL1-/- and MGL1-/- mice who received an anti-MGL1/2 blocking antibody both showed significantly increased endogenous FVIII activity compared with wild-type mice (P=0.036 and P<0.0001, respectively). MGL inhibition also prolonged the half-life of infused FVIII in FVIII-/- mice. To assess whether MGL plays a role in the clearance of free FVIII in a VWF-independent manner, in vivo clearance experiments were repeated in dual VWF-/-/FVIII-/- mice. Importantly, the rapid clearance of free FVIII in VWF-/-/FVIII-/- mice was significantly (P=0.012) prolonged in the presence of anti-MGL1/2 antibodies. Finally, endogenous plasma FVIII levels in VWF-/- mice were significantly increased following MGL inhibition (P=0.016). CONCLUSIONS: Cumulatively, these findings demonstrate that MGL plays an important role in regulating macrophage-mediated clearance of both VWF-bound FVIII and free-FVIII in vivo. We propose that this novel FVIII clearance pathway may be of particular clinical importance in patients with type 2N or type 3 Von Willebrand disease.

Physical activity, physical fitness and cardiometabolic risk amongst adults with moderate and severe haemophilia.

AIM: This study aimed to examine physical activity (PA), physical fitness and cardiometabolic risk amongst people with moderate and severe haemophilia (PwMSH). METHODS: The following domains were examined: PA (accelerometry); functional aerobic capacity (6-Minute Walk Test); grip strength (dynamometry); balance (One Leg Stand Test); body composition (anthropometry and bioimpedance analysis); blood pressure; arterial stiffness; and cardiometabolic disorders. RESULTS: A total of 53 PwMSH (44 years) and 33 controls (43 years; p = .679) were recruited. Compared to controls, PwMSH were significantly less active in moderate and vigorous PA parameters (all p < .05), and less physically fit indicated by 6-Minute Walk distance (p < .0005), grip strength (p = .040) and balance (p < .0005). PwMSH had higher rates of abdominal adiposity compared to controls measured by waist circumference indices (all p < .05). Resting blood pressure and arterial stiffness were not significantly different (p = .797 and .818, respectively). With respect to overall PA, World Health Organisation recommended targets for adults were achieved by the majority of both groups (haemophilia: 72.9% vs. controls: 90.0%; p = .069). Importantly, the number of PwMSH who achieved guideline recommended PA via longer, sustained bouts of moderate-vigorous PA was significantly lower compared to controls (18.8% vs. 56.7%; p = .001). Lastly, clinically diagnosed hypertension, insulin resistance and hyperlipidaemia were more prevalent amongst PwMSH compared to controls. CONCLUSION: Low levels of PA and physical fitness, and significant rates of abdominal adiposity and hypertension may collectively influence the risk and severity of various cardiometabolic and/or musculoskeletal health issues amongst ageing PwMSH. Personalised multi-disciplinary health interventions involving PA, dietary and health psychology input for PwMSH warrant future investigation.

A cross-sectional follow-up study of physical activity in adults with moderate and severe haemophilia.

AIM: To conduct a cross-sectional follow-up assessment of physical activity (PA) in people with moderate and severe haemophilia (PwMSH) from the Irish Personalised Approach to the Treatment of Haemophilia (iPATH) study. METHODS: Between June-December 2021, participants' PA was measured over one week using accelerometery, and was compared with their previously measured data from the original iPATH assessment. Self-awareness of PA and the impact of the Covid-19 pandemic on PA, pain, mobility and function were retrospectively examined using a survey. RESULTS: Of 30 participants who returned surveys [n = 19, severe (FVIII, <.01 IU/mL); n = 4, moderate (FVIII, .01-.05 IU/mL); n = 7, severe (FIX, <.01 IU/mL); age: 47 (36, 55) years], 28 completed accelerometery (follow-up time: 3 years). There were no significant differences in accelerometer PA (all p > .05), but achievement of World Health Organisation guidelines increased (67.9%-75.0%; p = .646). Increased self-awareness of PA was reported by 76.7%, and 66.7% reported desires to become more physically active. Compared to normal, most reported either no differences or lower levels of PA during lockdown restrictions. Self-reported PA increased for most when restrictions eased from April 2021 onwards. Beyond the pandemic, concerns included pain and access to exercise resources. CONCLUSION: Self-reported PA throughout the pandemic was variable, whilst there were no significant differences in objectively measured PA between assessment periods, despite reports of increased self-awareness and desires to be physically active at follow-up. Further qualitative research is needed to design personalised PA and health interventions, capturing perspectives of patients, their families, and multi-disciplinary haemophilia healthcare providers.

Plasma procurement and plasma product safety in light of the COVID-19 pandemic from the perspective of the plasma industry.

This review, written from the perspective of the plasma industry, discusses plasma procurement and plasma product safety in light of the COVID-19 pandemic. The COVID-19 pandemic impacted the whole world and, therefore, not unexpectedly, the pharmaceutical industry too. In spite of this, the plasma protein industry has continued to provide life saving therapies to critically ill patients. Moreover, companies have collected COVID convalescent plasma (CP) to support development of investigational therapies, for example, hyperimmune globulins to potentially treat SARS-CoV-2 infection, and collaborated with those collecting COVID CP for direct transfusion, which has been made available under emergency use in the United States. For plasma that is fractionated to become a therapy, general knowledge of coronaviruses and numerous new studies on the structure and function of SARS-CoV-2 provide reassurance that existing industry precautions, including donor selection, as well as virus inactivation and removal steps during the manufacturing process are sufficient to maintain the high standards of virus safety of plasma products. The pandemic also revealed the vulnerability and inadequacy of the current plasma ecosystem. There is a need for more plasma to be collected around the world to meet the growing need for safe and efficacious plasma-derived therapies. This requires outdated regulatory and policy restrictions to be realigned with current scientific evidence. More countries around the world should be in a position to contribute to global supply of plasma so that patients with life-threatening conditions - and often no alternative therapeutic solutions - have better access to care.

Biochemical and cellular markers differentiate recovered, in-line filtered plasma, and plasma obtained by apheresis methods.

BACKGROUND AND OBJECTIVES: Assessment of plasma quality often focuses on the common safety tests for minimizing the risk of transmitting blood-borne pathogens. Little attention is paid to the possible quality attributes that ensure a consistent biochemical composition of plasma for fractionation. We therefore investigated the suitability of selected biochemical and haematological attributes that could be used as markers of plasma quality obtained by different separation and pre-treatment procedures. MATERIAL AND METHODS: We characterized six plasma types, including source plasma, plasma recovered by classic means and in-line filtered plasma, by determining the analytical attributes protein content, coagulation factors and markers of coagulation, contact and complement activation. Residual cell content and cell-specific variables were also measured. RESULTS: We found relevant differences between the plasma types in complement activation, as indicated by C3a measurements, while thrombin antithrombin complex values and, to a minor extent, activated factor XII concentrations indicated only moderate differences in activation levels of coagulation and contact systems. The most striking differences, however, were detected in residual cell content and concentrations of the platelet-associated proteins, platelet factor 4 and ß-thromboglobulin. We showed that leucocyte reduction filters disrupt cells. This includes platelets, thereby releasing the platelet-associated proteins platelet factor 4 and ß-thromboglobulin, and leucocytes as demonstrated by the release of elastase from polymorphonuclear leucocytes. Furthermore, the filtration processing of whole blood can lead to activation of the complement system. CONCLUSION: Our results show that biochemical and cellular surrogate markers are valuable discriminators of plasma types.

Pain and functional disability amongst adults with moderate and severe haemophilia from the Irish personalised approach to the treatment of haemophilia (iPATH) study.

OBJECTIVES: To establish the prevalence of pain and functional disability in Irish adults with moderate and severe haemophilia, and to examine demographic and lifestyle influences. METHODS: Males ≥18 years with moderate or severe haemophilia participated. Pain and function were examined using the PROBE questionnaire. RESULTS: Of 49 participants [median age 44 (IQR 32, 52) years], most had severe haemophilia (Factor VIII = 30; Factor IX = 13) and were on regular prophylaxis (88%). Those with moderate haemophilia (Factor VIII = 5; Factor IX = 1) treated on demand (12%). Acute (72%) and chronic pain (71%), functional difficulties (58%), and analgesic requirements (92%) were prevalent. Age was significantly associated with more advanced haemophilic arthropathy (p = .002), chronic pain (p = .029) and functional difficulties (p = .036). Adults who reported chronic pain commenced prophylaxis significantly later in life [32 (20, 51) vs. 8 (1, 23) years; p = .004]. Physical activity was significantly lower in those with functional difficulties (p < .05). A disparity between self-perceived 'target joints' and clinically defined target joints was also identified (76% vs. 23%). CONCLUSION: Haemophilic arthropathy, pain and functional disability were prevalent amongst Irish adults with moderate and severe haemophilia. Age-dependent lifestyle, analgesic and treatment influences on pain and function warrant further investigation.

A systematic review of physical activity in people with haemophilia and its relationship with bleeding phenotype and treatment regimen.

INTRODUCTION: Although the measurement of physical activity (PA) amongst people with haemophilia (PWH) has become increasingly widespread in recent years, the relationship between PA and bleeding phenotype remains poorly understood. In addition, the influence of various treatment regimens on this relationship has not been defined. AIM: This review aimed to systematically assess the data that are available regarding PA levels amongst PWH, as well as the relationship between PA and bleeding. METHODS: A systematic search of the online databases EMBASE, Cochrane, MEDLINE Ovid, CINAHL and Web of Science was conducted by two independent reviewers. Quality assessment was undertaken using the AXIS Critical Appraisal Tool for Cross-sectional Studies and the STROBE checklist. RESULTS: Of 1902 sources identified overall, 36 articles were included. Low-to-moderate transparency of reporting and various sources of bias were identified. PA levels varied amongst heterogeneous samples of PWH. The relationship between PA and bleeds was inconclusive, although there was evidence that improvements in treatment over recent decades have appeared to enable PWH to become more physically active. CONCLUSION: Based upon the limited available evidence, the relationship between PA and bleeding phenotype in PWH remains unclear. However, with the development of improved prophylaxis treatment regimens in recent years, there is evidence that PA levels have increased, especially amongst people with severe haemophilia. The use of validated outcome measures of PA and more robust reporting of bleeds and treatment regimen are warranted in future research, especially in a rapidly evolving era of new treatments for PWH.

Biological mechanisms underlying inter-individual variation in factor VIII clearance in haemophilia.

Previous studies have highlighted marked inter-individual variations in factor VIII (FVIII) clearance between patients with haemophilia (PWH). The half-life of infused FVIII has been reported to vary from as little as 5.3 hours in some adult PWH, up to as long as 28.8 hours in other individuals. These differences in clearance kinetics have been consistently observed using a number of different plasma-derived and recombinant FVIII products. Furthermore, recent studies have demonstrated that half-life for extended half-life (EHL-) FVIII products also demonstrates significant inter-patient variation. Since time spent with FVIII trough levels <1% has been shown to be associated with increased bleeding risk in PWH on prophylaxis therapy, this variability in FVIII clearance clearly has major clinical significance. Recent studies have provided significant novel insights into the cellular basis underlying FVIII clearance pathways. In addition, accumulating data have shown that endogenous plasma VWF levels, ABO blood group and age, all play important roles in regulating FVIII half-life in PWH. Indeed, multiple regression analysis suggests that together these factors account for approximately 34% of the total inter-individual variation in FVIII clearance observed between subjects with severe haemophilia A. In this review, we consider these and other putative modulators of FVIII half-life, and discuss the biological mechanisms through which these factors impact upon FVIII clearance in vivo.

Evaluation of Factor VIII Polysialylation: Identification of a Longer-Acting Experimental Therapy in Mice and Monkeys.

Extended half-life (EHL) factor therapies are needed to reduce the burden of prophylaxis and improve treatment adherence in patients with hemophilia. BAX 826 is a novel polysialylated full-length recombinant factor VIII [polysialyic acid (PSA) rFVIII] with improved pharmacokinetics (PK), prolonged pharmacology, and maintained safety attributes to enable longer-acting rFVIII therapy. In factor VIII (FVIII)-deficient hemophilic mice, PSArFVIII showed a substantially higher mean residence time (>2-fold) and exposure (>3-fold), and prolonged efficacy in tail-bleeding experiments (48 vs. 30 hours) compared with unmodified recombinant FVIII (rFVIII), as well as a potentially favorable immunogenicity profile. Reduced binding to a scavenger receptor (low-density lipoprotein receptor-related protein 1) and von Willebrand factor (VWF) as well as a largely VWF-independent circulation time in mice provide a rationale for prolonged BAX 826 activity. The significantly improved PK profile versus rFVIII was confirmed in cynomolgus monkeys [mean residence time: 23.4 vs. 10.1 hours; exposure (area under the curve from time 0 to infinity): 206 vs. 48.2 IU/ml⋅h] and is in line with results from rodent studies. Finally, safety and toxicity evaluations did not indicate increased thrombogenic potential, and repeated administration of BAX 826 to monkeys and rats was well tolerated. The favorable profile and mechanism of this novel experimental therapeutic demonstrated all of the requirements for an EHL-rFVIII candidate, and thus BAX 826 was entered into clinical assessment for the treatment of hemophilia A. SIGNIFICANCE STATEMENT: Prolongation of FVIII half-life aims to reduce the burden of prophylaxis and improve treatment outcomes in patients with hemophilia. This study shows that polysialylation of PSArFVIII resulted in prolongations of rFVIII circulation time and procoagulant activity, together with a favorable nonclinical safety profile of the experimental therapeutic.

Sensitive and specific assessment of recombinant von Willebrand factor in platelet function analyzer.

BACKGROUND: Recombinant von Willebrand factor (rVWF), which was licensed in the United States in 2015, has the multimeric distribution of freshly secreted VWF with ultralarge (UL) and high molecular weight multimers (HMWM) from endothelial cells and megakaryocytes since it has never been exposed to ADAMTS13 or any other proteolytic enzyme. Measurement of closure time (CT) using the platelet function analyzer-200 (PFA-200) is highly sensitive to the presence of UL VWF multimers in added VWF concentrates. The PFA-200 is fully automated and can be used as a reliable point-of-care method to evaluate primary hemostasis. Although it is sensitive to presence of UL VWF multimers, there could be significant clinical utility when used to monitor rVWF replacement therapy. The ability to monitor and optimize the dosing of rVWF contributes to patient safety, especially in situations where the bleeding and thrombotic risk needs to be carefully balanced (e.g., cardiac assist device). OBJECTIVE: The aim of this in-vitro study was to demonstrate the detectability of rVWF spiked in VWF-deficient blood from patients with severe von Willebrand disease (VWD) with quantitative and functional pathologies using a functional testing device. We hypothesized that (1) whole blood samples from VWD patients spiked with rVWF would show a normalization in PFA-CT and (2) that a dose-response relationship could be demonstrated. METHODS AND RESULTS: We selected 12 patients diagnosed with VWD from our database. A therapeutic dose of rVWF product (1 IU/ml) was spiked in VWD patients´ whole blood samples and PFA-CTs were measured. Furthermore, we investigated PFA-CTs under incremental doses of rVWF (0.1, 0.2, and 0.5 IU/ml). The PFA-CTs were normalized in VWD patients´ whole blood samples spiked with rVWF. Additionally, incremental doses of rVWF resulted in a progressive and dose-dependent PFA-CT correction. CONCLUSION: Our in-vitro data indicate that the PFA-200 is a useful tool to detect rVWF. As the PFA-CT correction is dose dependent, the rVWF might be reliably monitored with a point-of-care analytical method during replacement therapy.

Issues complicating precision dosing for factor VIII prophylaxis.

We previously showed that personalizing prophylaxis on the basis of an individual's pharmacokinetic (PK) response to factor VIII (FVIII) infusion reduces joint and other bleeding events in patients with hemophilia A. We theorized that the FVIII assay used, FVIII product selected, and interpatient differences impact PK assessment and the ability to precisely dose prophylaxis. A comprehensive search of the literature for articles published from January 2004 to September 2017 was performed to identify the variables associated with these three domains. Collectively, product- and patient-related assay discrepancies, variability among plasma-derived and unmodified and modified recombinant FVIII products, and interpatient differences in the response to FVIII infusions are obstacles to precision prophylactic dosing. Stringent laboratory quality assurance programs and proficiency testing to improve the accuracy of FVIII measurement, the widespread use of PK assessment to fine-tune FVIII dosing, and new research to identify patient characteristics and other contributors to bleeding risk and complication development are essential to optimizing outcomes for patients with hemophilia A receiving FVIII prophylaxis.

Low ADAMTS13 activity is associated with an increased risk of ischemic stroke.

ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin motif repeats 13) has antithrombotic properties because it cleaves von Willebrand factor (VWF) in smaller, less active multimers. The aim of our study was to investigate prospectively the association between ADAMTS13 activity and ischemic stroke. We included 5941 individuals ≥55 years without a history of stroke or transient ischemic attack (TIA) of the Rotterdam Study, a population-based cohort study. ADAMTS13 activity was measured at inclusion with the FRETS-VWF73 assay and VWF antigen (VWF:Ag) levels by enzyme-linked immunosorbent assay. We assessed the association among ADAMTS13 activity, VWF:Ag levels, and ischemic stroke by Cox proportional hazard analysis. The added value of ADAMTS13 activity above the traditional risk factors for ischemic stroke risk prediction was examined by the C-statistic and the net reclassification improvement index (NRI). All individuals were followed for incident stroke or TIA. Over a median follow-up time of 10.7 years (56,403 total person-years), 461 participants had a stroke, 306 of which were ischemic. After adjustment for cardiovascular risk factors, individuals with ADAMTS13 activity in the lowest quartile had a higher risk of ischemic stroke (absolute risk, 7.3%) than did those in the reference highest quartile (absolute risk, 3.8%; hazard ratio, 1.65; 95% confidence interval [CI], 1.16-2.32). Adding ADAMTS13 to the model in prediction of ischemic stroke, increased the C-statistic by 0.013 (P = .003) and provided 0.058 (95% CI, -0.002 to 0.119) NRI. Low ADAMTS13 activity is associated with the risk of ischemic stroke and improves the accuracy of risk predictions for ischemic stroke beyond traditional risk factors.

Shear-Dependent Interactions of von Willebrand Factor with Factor VIII and Protease ADAMTS 13 Demonstrated at a Single Molecule Level by Atomic Force Microscopy.

Vital functions of mammals are only possible due to the behavior of blood to coagulate most efficiently in vessels with particularly high wall shear rates. This is caused by the functional changes of the von Willebrand Factor (VWF), which mediates coagulation of blood platelets (primary hemostasis) especially when it is stretched under shear stress. Our data show that shear stretching also affects other functions of VWF: Using a customized device to simulate shear conditions and to conserve the VWF molecules in their unstable, elongated conformation, we visualize at single molecule level by AFM that VWF is preferentially cleaved by the protease ADAMTS13 at higher shear rates. In contrast to this high shear-rate-selective behavior, VWF binds FVIII more effectively only below a critical shear rate of ∼30.000 s(-1), indicating that under harsh shear conditions FVIII is released from its carrier protein. This may be required to facilitate delivery of FVIII locally to promote secondary hemostasis.

Development, Validation, and Application of a Novel Ligand-Binding Assay to Selectively Measure PEGylated Recombinant Human Coagulation Factor VIII (BAX 855).

BAX 855 is a PEGylated recombinant factor VIII preparation that showed prolonged circulatory half-life in nonclinical and clinical studies. This paper describes the development, validation, and application of a novel ligand-binding assay (LBA) to selectively measure BAX 855 in plasma. The LBA is based on PEG-specific capture of BAX 855, followed by immunological factor VIII (FVIII)-specific detection of the antibody-bound BAX 855. This assay principle enabled sensitive measurement of BAX 855 down to the low nanomolar range without interference from non-PEGylated FVIII as demonstrated by validation data for plasma from animals typically used for nonclinical characterization of FVIII. The selectivity of an in-house-developed anti-PEG and a commercially available preparation, shown by competition studies to primarily target the terminating methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, this new LBA adds to the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. The feasibility and convenience of using this method was demonstrated during extensive nonclinical characterization of BAX 855.

Pharmacokinetics and safety of a novel recombinant human von Willebrand factor manufactured with a plasma-free method: a prospective clinical trial.

Safety and pharmacokinetics (PK) of recombinant von Willebrand factor (rVWF) combined at a fixed ratio with recombinant factor VIII (rFVIII) were investigated in 32 subjects with type 3 or severe type 1 von Willebrand disease (VWD) in a prospective phase 1, multicenter, randomized clinical trial. rVWF was well tolerated and no thrombotic events, inhibitors, or serious adverse events were observed. The PK of rVWF ristocetin cofactor activity, VWF antigen, and collagen-binding activity were similar to those of the comparator plasma-derived (pd) VWF-pdFVIII. In vivo cleavage of ultra-large molecular-weight rVWF multimers by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; the endogenous VWF protease) and generation of characteristic satellite bands were demonstrated. In 2 subjects with specific nonneutralizing anti-VWF-binding antibodies already detectable before rVWF infusion, a reduction in VWF multimers and VWF activity was observed. Stabilization of endogenous FVIII was enhanced following post-rVWF-rFVIII infusion as shown by the difference in area under the plasma concentration curve compared with pdVWF-pdFVIII (AUC0-∞) (P < .01). These data support the concept of administering rVWF alone once a therapeutic level of endogenous FVIII is achieved.

A Flow-Cytometry-Based Approach to Facilitate Quantification, Size Estimation and Characterization of Sub-visible Particles in Protein Solutions.

PURPOSE: Sub-visible particles were shown to facilitate unwanted immunogenicity of protein therapeutics. To understand the root cause of this phenomenon, a comprehensive analysis of these particles is required. We aimed at establishing a flow-cytometry-based technology to analyze the amount, size distribution and nature of sub-visible particles in protein solutions. METHODS: We adjusted the settings of a BD FACS Canto II by tuning the forward scatter and the side scatter detectors and by using size calibration beads to facilitate the analysis of particles with sizes below 1 µM. We applied a combination of Bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt) and DCVJ (9-(2,2-dicyanovinyl)julolidine) to identify specific characteristics of sub-visible particles. RESULTS: The FACS technology allows the analysis of particles between 0.75 and 10 µm in size, requiring relatively small sample volumes. Protein containing particles can be distinguished from non-protein particles and cross-ß-sheet structures contained in protein particles can be identified. CONCLUSIONS: The FACS technology provides robust and reproducible results with respect to number, size distribution and specific characteristics of sub-visible particles between 0.75 and 10 µm in size. Our data for number and size distribution of particles is in good agreement with results obtained with the state-of-the-art technology micro-flow imaging.

PEGylated Biopharmaceuticals: Current Experience and Considerations for Nonclinical Development.

PEGylation (the covalent binding of one or more polyethylene glycol molecules to another molecule) is a technology frequently used to improve the half-life and other pharmaceutical or pharmacological properties of proteins, peptides, and aptamers. To date, 11 PEGylated biopharmaceuticals have been approved and there is indication that many more are in nonclinical or clinical development. Adverse effects seen with those in toxicology studies are mostly related to the active part of the drug molecule and not to polyethylene glycol (PEG). In 5 of the 11 approved and 10 of the 17 PEGylated biopharmaceuticals in a 2013 industry survey presented here, cellular vacuolation is histologically observed in toxicology studies in certain organs and tissues. No other effects attributed to PEG alone have been reported. Importantly, vacuolation, which occurs mainly in phagocytes, has not been linked with changes in organ function in these toxicology studies. This article was authored through collaborative efforts of industry toxicologists/nonclinical scientists to address the nonclinical safety of large PEG molecules (>10 kilo Dalton) in PEGylated biopharmaceuticals. The impact of the PEG molecule on overall nonclinical safety assessments of PEGylated biopharmaceuticals is discussed, and toxicological information from a 2013 industry survey on PEGylated biopharmaceuticals under development is summarized. Results will contribute to the database of toxicological information publicly available for PEG and PEGylated biopharmaceuticals.

Ca(2+) concentration-dependent conformational change of FVIII B-domain observed by atomic force microscopy.

FVIII is a multi-domain protein organized in a heavy and a light chain, and a B-domain whose biological function is still a matter of debate. The 3D structure of a B-domain-deleted FVIII variant has been determined by X-ray crystallography, leaving unexplained the functional nature of the flexible B-domain which could play an important role in the structure-function relationship since it is removed during the activation process. To obtain clues on the function of the B-domain, the morphology of full-length FVIII and its isolated domains was determined in the absence or presence of Ca(2+). Recombinant full-length FVIII, the purified heavy chain, light chain and B-domain as well as B-domain-deleted rFVIII were analysed in buffers of different Ca(2+) concentrations by atomic force microscopy. In the absence of Ca(2+), FVIII appeared as a globular molecule, whereas at high amounts of Ca(2+) up to 50-nm long tail structures emerged. These tails could be identified as unravelled B-domains, as images of isolated B-domains showed the same morphology and heavy chains which include the B-domain were also rich of tails, whereas the isolated light chains and B-domain-deleted FVIII lacked any deviation from a globular shape. The images further suggested that the B-domain interacts with the light chain particularly at low Ca(2+) concentrations. Our results show a Ca(2+)-regulated conformational change of the B-domain in the context of full-length rFVIII. As the B-domain tightly associated with the core of the FVIII molecule under low Ca(2+)-concentrations, a stabilizing function on FVIII under non-activating conditions may be proposed.