Congenital fibrinogen disorders (CFDs) are a heterogeneous group of rare congenital quantitative and/or qualitative fibrinogen deficiencies. The spectrum of molecular anomalies is broad, leading to several subtypes of fibrinogen disorders (ie, afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia). Pregnancy in women with CFDs is a high-risk clinical situation, with an increased tendency for miscarriages, bleeding, and thrombosis. Even though it is well established that management of such pregnancies requires a multidisciplinary approach involving specialists (hematologists and maternal/fetal medicine experts with expertise in the management of inherited bleeding disorders), specific guidelines are lacking. In this International Society on Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee communication, we aim to propose an expert consensus opinion with literature evidence where available on the strategy for management of pregnancy, delivery, and puerperium in CFDs.

Chromogenic assay discrepancies were reported at General European Official Medicines Control Laboratories Network (GEON) meetings by laboratories testing FVIII-products. The objectives of the present investigation were to carry out a controlled collaborative study to examine these reports and to delineate the reasons for these discrepancies by assessing affected and unaffected FVIII products. The laboratories followed a strict study protocol, which included assessing their own individual observed factor X (FX) activation times, i.e. the time to reach 50% of maximal FX activation (T1/2), for each chromogenic kit. This measurement was used, in parallel with the kit manufacturers' prescribed FX activation times, to assess the performance of the chromogenic potency assays on FVIII test products. This study confirmed a significant discrepancy between Coatest® and Coamatic® kits and between Siemens and Coamatic® kits when the kit manufacturers' prescribed T1/2 incubation times were followed. Coamatic® kits tended to produce higher potencies than the Coatest® or Siemens kits. Furthermore, FX activation assays revealed marked differences between individual laboratories for all three chromogenic kits in the observed T1/2 incubation times, which also did not correspond to the prescribed T1/2 incubation times. The resulting differences in potency between kits, in some cases, were significantly reduced when using the actual observed T1/2 incubation times instead of the prescribed T1/2 incubation times. The study showed that FVIII potency discrepancies can occur between chromogenic kits. To compensate for this, laboratories should ideally perform FX activation curves for each new chromogenic kit in order to determine the correct observed T1/2 incubation times, which can then be used to determine FVIII potencies in therapeutic concentrates.

Allosteric disulfide bonds permit highly responsive, transient 'switch-like' properties that are ideal for processes like coagulation and inflammation that require rapid and localised responses to damage or injury. Haemophilia A (HA) is a rare bleeding disorder managed with exogenous coagulation factor(F) VIII products. FVIII has eight disulfide bonds and is known to be redox labile, but it is not known how reduction/oxidation affects the structure-function relationship, or its immunogenicity-a serious complication for 30% severe HA patients. Understanding how redox-mediated changes influence FVIII can inform molecular engineering strategies aimed at improving activity and stability, and reducing immunogenicity. FVIII is a challenging molecule to work with owing to its poor expression and instability so, in a proof-of-concept study, we used molecular dynamics (MD) to identify which disulfide bonds were most likely to be reduced and how this would affect structure/function; results were then experimentally verified. MD identified Cys1899-Cys1903 disulfide as the most likely to undergo reduction based on energy and proximity criteria. Further MD suggested this reduction led to a more open conformation. Here we present our findings and highlight the value of MD approaches.

The European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for human coagulation Factor VIII (FVIII) Concentrate is used as working standard for potency determination of human coagulation FVIII preparations by chromogenic assay. BRP batch 5 was established in 2015 and its stocks were running low. Therefore, the European Directorate for the Quality of Medicines & HealthCare (EDQM) initiated a project (BSP156) for the calibration of a replacement batch. The potency of BRP batch 6 was assigned during an international collaborative study involving 16 laboratories worldwide, with reference to the WHO 8th International Standard (IS) and BRP batch 5. Participants were instructed to perform 3 independent FVIII potency assays following their own routine validated methods for the chromogenic assay, which is the assay prescribed by the Ph. Eur. As an outcome of the study, Ph. Eur. human coagulation FVIII Concentrate BRP batch 6 was assigned a consensus potency of 9.9 IU/ampoule for the chromogenic assay. The Ph. Eur. BRP batch 6 is a freeze-dried, plasma-derived concentrate. Based on accelerated degradation studies, the stability of the material is suitable for a reference preparation. The Ph. Eur. BRP batch 6 was adopted at the 167th session of the Ph. Eur. Commission in June 2020 and is available from the EDQM under product code H0920000.

The coagulation factors (F)V and VIII are homologous proteins that support hemostasis through their regulation of FX activity. Hemophilia A (HA) patients have reduced FVIII activity and a prolonged bleeding time that is corrected through the administration of exogenous FVIII. Around one‐third of severe HA patients develop FVIII neutralizing antibodies, known as “inhibitors,” which neutralize FVIII activity and preclude them from further FVIII therapy.

BACKGROUND:Factor XIII (FXIII)-B subunit measurements are required for the diagnosis and characterization of the type of FXIII deficiency. Furthermore, therapy for FXIII-A deficiency with recombinant FXIII (rFXIII-A) relies on available FXIII-B.OBJECTIVE:To carry out a collaborative study to calibrate and assign value to the current WHO 1st International Standard (IS) FXIII Plasma for Total FXIII-B subunit, relative to locally collected normal plasma pools.METHODS:Laboratories were instructed to use a validated method (specific ELISA antibodies provided) for assessment of Total FXIII-B subunit antigen potency. All laboratories used this method with one laboratory using an additional in-house method. Nine data sets were received from seven laboratories (37 assays in total), which provided a total of 35 valid estimates for this new assignment. Total FXIII-B subunit estimates were calculated relative to locally collected normal plasma pools, using an arbitrary value of 1.00 unit of Total FXIII-B subunit per ml, for each pool.RESULTS:Combination of results produced an overall mean of 0.98 units/mL with an inter-laboratory variability (geometric coefficients of variation - GCV%) of 18.3% [95% confidence interval: 0.86-1.11]. Real-time and bench stability studies indicated good stability and preservation of the FXIII-B subunit analyte in the WHO 1st IS FXIII Plasma (02/206).CONCLUSION:Following agreement by study participants, ISTH/SSC Experts, WHO-ISTH Liaison Group and the SSC Board, the WHO/ECBS established the current WHO 1st IS Factor XIII plasma (NIBSC code 02/206) by additionally assigning it with a Total FXIII-B subunit antigen value of 0.98 IU/ampoule, in October 2019.

The development of anti-drug antibodies (ADAs) is a serious outcome of treatment strategies involving biological medicines. Coagulation factor VIII (FVIII) is used to treat haemophilia A patients, but its immunogenicity precludes a third of severe haemophiliac patients from receiving this treatment. The availability of patient-derived anti-drug antibodies can help us better understand drug immunogenicity and identify ways to overcome it. Thus, there were two aims to this work: (i) to develop and characterise a panel of recombinant, patient-derived, monoclonal antibodies covering a range of FVIII epitopes with varying potencies, kinetics and mechanism of action, and (ii) to demonstrate their applicability to assay development, evaluation of FVIII molecules and basic research. For the first objective we used recombinant antibodies to develop a rapid, sensitive, flexible and reproducible ex vivo assay that recapitulates inhibitor patient blood using blood from healthy volunteers. We also demonstrate how the panel can provide important information about the efficacy of FVIII products and reagents without the need for patient or animal material. These materials can be used as experimental exemplars or controls, as well as tools for rational, hypothesis-driven research and assay development in relation to FVIII immunogenicity and FVIII-related products.

During the freezing step of a typical freeze drying process, the temperature at which nucleation is induced is generally stochastically distributed, resulting in undesired within-batch heterogeneity. Controlled nucleation techniques have been developed to address this problem; these make it possible to trigger the formation of ice crystals at the same time and temperature in all the batch. Here, the controlled nucleation technique known as vacuum induced surface freezing is compared to spontaneous freezing for the freeze drying of human plasma, a highly concentrated system commonly stored in a dried state. The potency of Factor VIII (FVIII), a sensitive, labile protein present in plasma, and the reconstitution time of the dried cakes are evaluated immediately after freeze drying, and after 1, 3, 6 or 9 months storage at different degradation temperatures. We show that the application of controlled nucleation significantly reduces the reconstitution time and in addition helps to improve FVIII stability.

The Second Meeting of the National Control Laboratories for Vaccines and Biologicals in the Western Pacific, was jointly organized by the National Institute of Food and Drug Safety Evaluation of the Ministry of Food and Drug Safety in the Republic of Korea, and by the World Health Organization Regional Office for the Western Pacific. In the National Lot Release Systems session countries including Canada, China, Japan, Malaysia, Vietnam, and the Republic of Korea, all shared information on their current Lot Release Systems, including current practices and developments in risk-based official lot release of vaccines. In the session on Quality Control of Blood Products, experts from the National Institute for Biological Standards and Control shared quality control and research results for; blood coagulation factor VIII products, and the measurement of procoagulant activity in immunoglobulin products. Representatives from Japan proposed a regional collaborative study to test aggregated immunoglobulin free from complement activity. A cell-based Japanese encephalitis vaccine potency assay was proposed by representatives from Korea and they also called for voluntary participation of other National Control Laboratories in a collaborative study, on the first Korean Gloydius anti-venom standard. Participants agreed in general to continue communicating, and coordinate presentation of the study results.

The European Pharmacopoeia Biological Reference Preparation (Ph. Eur. BRP) for Factor VIII Concentrate batch 5 was established through a collaborative study involving 14 laboratories organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe) to be used as working standard for potency determination of human coagulation Factor VIII (FVIII) preparations. The potency of the BRP batch 5 was assigned with reference to the WHO 8th International Standard (IS) for FVIII Concentrate and the BRP batch 4. Participants were instructed to perform 3 independent Factor VIII potency assays following their own routine validated methods by the chromogenic assay as it is the assay prescribed by the European Pharmacopoeia. This publication reports the results obtained during the study. The consensus potency, 9.9 IU/ampoule (n = 14) when assessed against both standards, with inter-laboratory geometric coefficients of variation (GCV) of 3.2 % and 1.9 % against the WHO 8th IS and the BRP batch 4 respectively, was consistent with the expected value. The Ph. Eur. BRP batch 5 is a freeze-dried, plasma-derived concentrate. Based on accelerated degradation studies, the stability of the material is suitable as a reference preparation. The Ph. Eur. BRP batch 5 was adopted at the 151st session of the European Pharmacopoeia Commission in March 2015 and is available from the EDQM.

Residual moisture is a key quality control parameter for lyophilized biologicals, as high moisture can correlate with poor stability.Coulometric Karl Fischer titration is the most widely used technology to determine residual water content; some chemicals are known to cause problems with Karl Fischer titration, but these chemicals do not usually occur in biologics.Three biological samples, of fibrinogen, heparin and Haemophilus influenza b polysaccharide, have caused particular issue in our hands by routine Karl Fischer analysis, illustrating different limitations with this method.The use of thermogravimetric analysis, with evolved gas mass spectrometric monitoring, is described here as a successful alternative for moisture analysis in these materials.

Following the reporting of a positive association between fibrinogen levels and risk of cardiovascular diseases or risk of bleeding (e.g. DIC) when screening for possible acquired or congenital deficiency 1-3, a number of commercial kits had emerged onto the market for measurement of physiological fibrinogen as a marker for such diseases. The variability of reference plasma standards provided in these kits, 4, 5 necessitated the development of the 1st World Health Organization (WHO) International Standard (IS) for Fibrinogen Plasma 6 in order to calibrate the secondary plasma standards provided in these commercial kits and to standardize different fibrinogen assay methods. The 2nd IS for Fibrinogen Plasma (98/612) was established by the WHO in November 1999 with an assigned potency of 2.2 mg per ampoule 7 for thombin clottable protein where calibration was achieved using primarily Clauss assays against the WHO 1st IS for Fibrinogen Plasma (89/644) 6. Due to low stock numbers, replacement of the 2nd IS was necessary and this study describes the value assignment of the replacement preparation, with the primary objective of a robust transference of the thrombin clottable fibrinogen unitage between the 2nd IS and the proposed replacement 3rd IS. The candidate material (coded 09/264) evaluated as a potential replacement was a virus-inactivated fresh-frozen plasma product (citrated plasma with solvent-detergent treatment using Octoxynol and TNBP for 4 h at 30 °C). For the sake of harmonization and continuity of the unitage, assays on the candidate material were carried out using thrombin-clottable protein (functional fibrinogen) assays, relative to the 2nd IS 7, in an international collaborative study involving 21 laboratories. All laboratories were instructed to use their routine validated methods and to reconstitute all test preparations with 1 mL of distilled water. Laboratories returned data from Clauss assays (22 datasets) or subsequent assay methods following clot removal (CLOTr) (e.g. Kjeldhal or absorbance assays, two datasets), or PT derived assays (one dataset). In addition, three laboratories carried out immunological assays (e.g. ELISAs, two datasets) or used a Nephelometry method (one dataset). Several laboratories used more than one assay method (laboratories 8, 10, 14 and 20) or more than one variation of the same method (laboratories 7 and 18). Laboratories 7 and 18 each performed two different Clauss assays, with different kits and different thrombin concentrations, respectively. They were analyzed separately and coded 7A, 7B and 18A, 18B, respectively. Potency estimates were calculated from raw assay data according to parallel line bioassay principles 8. Mean results from individual laboratories were combined using unweighted geometric means and the intra- and inter-laboratory variability was expressed as the geometric coefficient of variation (GCV%) 9. Table 1 summarizes the overall mean estimates and inter-laboratory variability from the collaborative study. Geometric mean = 2.66 (n = 22) Inter-laboratory GCV = 3.2% Combined geometric mean = 2.67 (n = 24) Combined inter-laboratory GCV = 3.3% (excluding immunological assays† † Immunological assays. *Statistical outliers. and statistical outliers* † Immunological assays. *Statistical outliers. ) Intra-laboratory GCVs were found to range from 0.7 to 7.3% for Clauss assays, from 2.5 to 6.7% for CLOTr methods and from 6.7 to 12.4% for ELISAs, with GCVs from all the laboratories below 8% (except for one laboratory). There was very good agreement regarding potencies between laboratories using each of the different methods when assaying the candidate sample (09/264), relative to the 2nd IS (see Fig. 1), with GCV of 3.2% and mean potency of 2.66 mg mL−1 by Clauss assay (n = 22), potency values of 2.86 and 2.69 mg mL−1 by the CLOTr method, and potency values of 2.39 and 2.73 mg mL−1 by ELISA. A combined mean potency value of 2.67 mg mL−1 was obtained for the candidate sample (09/264), with a combined GCV value of 3.3% (n = 24). Results from immunological assays were not included in the combined overall means as they do not represent the functional activity of fibrinogen. It is important to emphasize that the number of datasets for each of the non-Clauss methods was very low (n = 2) and that overall combined potencies primarily comprised data from Clauss assays. Overall, the candidate sample (09/264) is found to be a suitable replacement for the current 2nd IS for Fibrinogen Plasma due to an excellent agreement of mean values obtained for thrombin clottable protein methods (< 4% discrepancy between the Clauss assay and the CLOTr method), low overall inter-laboratory variability for combined estimates (GCV = 3.3%) and good predicted stability following accelerated degradation stability studies (data not shown) 10. The validity of assays relative to the 2nd IS Fibrinogen Plasma (98/612) included in the study, confirms that the candidate sample (09/264) is suitable to serve as the 3rd IS Fibrinogen Plasma and also ensures excellent continuity of the plasma unit from the current 2nd IS. The normal procedure when calibrating International Standards is to take the mean potency of the proposed standard, by all valid estimates, vs. the previous standard. This is the best way of ensuring continuity between old and new standards. An accelerated degradation study carried out on the previous WHO 2nd IS Fibrinogen Plasma (98/612) in June 2011, showed that this standard is extremely stable when stored at −20 °C, with no measureable loss after 12 years of storage. The stability data for the WHO 2nd IS Fibrinogen Plasma (98/612) therefore validates its use as a reference for the calibration of the proposed WHO 3rd International Standard for Fibrinogen Plasma. In addition to the 21 participants (See Appendix), this study was reviewed by 28 further experts in the field, all of whom approved and endorsed this standard as a suitable replacement. It was therefore proposed that the candidate preparation (NIBSC code 09/264) be accepted as the 3rd International Standard for Fibrinogen Plasma with an assigned potency of 2.7 mg per ampoule. This proposal was endorsed by the ISTH/SSC and preparation 09/264 was established by the WHO in November 2011 10. S. Raut designed the study, analyzed the study data and wrote and reviewed the manuscript. S. Daniels performed provisional research, analyzed the study data and reviewed the manuscript. M. Hamill and A. B. Heath statistically analyzed the study data and reviewed the manuscript. The contributions of all the participants in the study are gratefully acknowledged. We are grateful to our colleagues in the Standards Division, NIBSC, for ampouling and processing the candidate and trial preparations and for the dispatch of collaborative study samples to participants. We are grateful to Kedrion S.p.A. (Italy) and Octapharma (Austria) for their kind donation of materials for the study. We would further like to thank the ISTH/SSC Fibrinogen and Factor XIII Subcommittee for their guidance and all the experts who reviewed this study. The authors state that they have no conflict of interests.

SummaryFactor‐Eight‐Inhibitor‐Bypassing‐Activity (FEIBA) is a bypassing‐agent used to control spontaneous bleeding or cover surgical interventions in Haemophiliacs who develop neutralizing antibodies against FVIII/FIX. The market lot‐release of FEIBA is dependent on specific clot‐based assays, carried out by both the manufacturer and regulatory authorities, relative to manufacturer's in‐house standards, which are produced on a small‐scale and are replaced frequently. We sought to standardize the FEIBA assay by developing a FEIBA primary standard which would be internationally available in sufficiently large quantities, with a predicted lifetime of many years. A collaborative study involving the manufacturer and three regulatory authorities, was carried out in which a candidate material, sample B (06/172), was calibrated by assays relative to the manufacturer's in‐house FEIBA standards (C and D). All laboratories used their routine validated methods (16 APTT‐assays, 8 ACTIN‐FS‐assays and 27 DAPTTIN‐assays). Intra‐laboratory geometric coefficients of variation (GCVs) for candidate B ranged from 3% to 29% (GCVs <9% from majority of labs). Assessment of inter‐laboratory variability gave overall GCV values of 6.9% and 4.4% relative to standards C and D, respectively, for all methods. There was good agreement in potency estimation between laboratories using each of the three methods, with the overall potencies by the three methods differing by less than 10% of the overall mean, giving an overall combined potency of 28.0 units per ampoule. All participants agreed that candidate B (06/172) be established as the 1st NIBSC Working Standard for FEIBA with an assigned potency of 28.0 units per ampoule, based on combined results for both methods, relative to either standard C or D.