Discrepancies in potency assessment of recombinant FVIII concentrates

Summary. Results of assays of recombinant FVIII concentrates have been reviewed over a 10-year period. Initially there was wide variability between laboratories but this was minimised by the development of standardised assay methodology, in particular the use of haemophilic plasma for pre-dilution and 1% albumin in assay buffers. Using this standardised methodology and concentrate standards, there were no major diferences in potency between one-stage, two-stage, and chromogenic assays on the two full-length recombinant FVIII concentrates. However, using a plasma standard, the chromogenic method gave much higher potencies than the one-stage method on the same concentrates, and this explains a similar discrepancy found in patients' post-infusion samples after injection of recombinant concentrates. It is suggested that concentrate standards be used for such post-infusion samples in order to minimise this discrepancy.     

Pharmacokinetics of plasma‐derived vs. recombinant FVIII concentrates: a comparative study

Only very few pharmacokinetic (PK) studies comparing plasma derived FVIII (pd‐FVIII) against recombinant FVIII (rFVIII) concentrates are available. The studies have been generally conducted to demonstrate the bioequivalence of a new product with an old one. The switch from a plasma‐derived FVIII (pd‐FVIII) to a rFVIII concentrate is a good moment to enrol the patients in a comparative PK study. To achieve information on the PK characteristics of two different classes of FVIII concentrates, according to two different designs: a 10 FVIII concentration/time point design and a reduced 4‐point design. A single dose PK comparing pd‐ and rFVIII concentrates has been performed in four Haemophilia Centres of Italy. Seventeen haemophilia A patients underwent two subsequent single dose PK studies at the moment of switching. Two‐compartment‐ and Non‐compartment‐analysis did not show significant differences between the outcomes of PK of pd‐FVIII and rFVIII, due to inter‐patient variability. In vivo recovery (IVR) of rFVIII was slightly higher than that of pd‐FVIII and rFVIII/pd‐FVIII AUC ratio was 1.37 in 11/17 patients. The difference is only due to the initial distribution phase because after the first 10 h from the end of the infusion, the two decay curves are overlapping. The elimination half‐life of the concentrates was very similar even though a complete bioequivalence was not demonstrated because of a higher AUC of rFVIII concentrates, limited to the distribution phase. The higher C_max and IVR of rFVIII may be due to the presence of heterodimers activated forms of the recombinant molecules.     

High incidence of anti-FVIII antibodies against non-coagulant epitopes in haemophilia A patients: a possible role for the half-life of transfused FVIII

The occurrence of antibodies (Abs) capable of inhibiting factor VIII (FVIII) coagulant activity is a severe complication in haemophilia A, leading to the inhibition of transfused FVIII activity. It is not known whether, or to what extent, post-transfusion antibodies may also arise against non-coagulant epitopes. Therefore we set up a system capable, in theory, to detect all the FVIII-induced antibodies by use of an enzyme-linked immunoassorbent assay (ELISA) based on coating human recombinant FVIII onto polystyrene microtitre plates. Serum samples from 23 patients affected by haemophilia A of different gravity (22 referred to our Centre and one to the Bari Centre) were analysed. Although only one patient was positive at Bethesda assay, the presence of antibodies in ELISA was detected in 39% of patients in variable degrees; transfusion with FVIII was found to induce a raise in antibody titre, arguing in favour of the specificity of the phenomenon. The clinical relevance of these non-inhibitory antibodies was evaluated in three patients; although half-life did not show any change in the patients without or with low amount of antibodies, FVIII clearance was found enhanced in the patient displaying high titre antibodies. We propose detection of anti-FVIII antibodies by ELISA when routinely assessing haemophilia A patients.     

VWF/FVIII concentrates in high-risk immunotolerance: the RESIST study

Summary.  Immune tolerance induction (ITI), through the regular infusion of coagulation factor concentrates over a time period ranging from 1 to more than 24 months, is successful in about 75% of high responders. Among the issues of ITI treatment that are still open, the choice of the product to use is one of the most difficult. In fact, common practice is to start with the same product that induced the inhibitory response, but recent findings indicated that plasma-derived products containing large amounts of von Willebrand factor (VWF) can play a positive role. Two retrospective cohorts in Germany and in France and one prospective cohort have shown a high rate of success when VWF/factor VIII (FVIII) products are used to induce ITI. For these reasons, two prospective studies have been planned to complement the international ITI study already started: an observational study in patients who had already experienced a failure with a VWF-free FVIII concentrate, called RESIST_exp (experienced); a randomized, controlled study in patients who have never tried an ITI treatment before and at high risk to fail, called RESIST_naïve (naïve).     

Long-term surveillance for human anti-murine antibodies of a group of haemophiliacs treated only with immunoaffinity-purified FVIII concentrates

An immunoaffinity purified, solvent/detergent virally inactivated factor VIII (FVIII) concentrate (Hemofil® M) has been in clinical use in persons with haemophilia A since March 1987 and under clinical trial prior to licencing in the USA, Europe and Japan.
The specification set and consistently met for the monoclonal antibody (mAb) content of the final product is 0.1 ng/IU FVIII. This level was considered non-immunogenic, based upon animal studies, recommendations of the European PHarmacopoeia with ovalbumin contamination of influenza vaccine and also the experience during clinical research studies using aAb for diagnostic or other clinical purpose as reported in the medical literature.
This research communication documents the surveillance for human anti-murine antibodies (HAMA) in 13 patients with serve haemophilia A. These patients have been treated with a large amount of product (mean 14,026 IU), numerous different lots of Hemofil M (total 45; mean 17) over a substantial period of time (mean 28 months).
The data generated from this group of 13 patients before and during therapeutic Hemofil M administration failed to show the development of any HAMA response at any timepoint. Therefore Hemofil M prepared by mAb resin immunoaffinity column chromatography under the currently set specifications for mAb contaminant of 0.1 ng/IU FVIII or less is a safe therapeutic agent in the management and prevention of bleeding episodes in persons with haemophilia A.     

New-generation recombinant factor concentrates: bridge to gene therapy

Despite our best efforts to deceive the immune system, outwit pathogens, and improve upon the design of nature, there continues to be a need to improve the margin of safety of treatment for those with bleeding disorders. The current approach includes: (1) recombinant factor concentrates free of added proteins; (2) 'designer' factor molecules that enhance function and reduce immunogenicity; and (3) modulation of the immune system to suppress immune response in those who develop inhibitors. The hope is that through advances in our understanding of the coagulation and immune systems, treatment of haemophilia in the new millennium will be safer and less immunogenic. Currently available recombinant clotting factor concentrates include those produced: (1) with pasteurized human serum albumin in the cell culture medium as a stabilizer; (2) with bovine serum proteins in the cell culture medium; and (3) free of plasma derivatives. To the extent that current recombinant clotting factor concentrates contain even trace amounts of human or animal protein, there is continuing potential for transmission of nonenveloped viruses, including hepatitis A and parvovirus, and the theoretical potential for transmission of relatively unknown agents, such as prions (Creutzfeldt–Jakob disease or its variant). Second-generation recombinant factor concentrates that do not use human albumin as a stabilizer are currently in clinical trials, and third-generation recombinant factor concentrates currently in development take advantage of new strategies to achieve a 'protein-free' cell culture, purification, and final formulation. It is likely that improvement in safety and reduction in immunogenicity will require modification not only of antigenic structure but also of the immune response to coagulation proteins.     

Phospholipid binding of factor VIII in different therapeutic concentrates

Binding to anionic phospholipid (PL) is essential for the biological function of factor VIII (FVIII). We have developed a method to study the level of PL binding of FVIII in a variety of therapeutic concentrates, using the BIACORETM system which utilizes the Surface Plasmon Resonance (SPR) phenomenon. A HPA sensor chip was employed on to which synthetic phospholipid unilamellar vesicles were adsorbed to form a 3:1 phosphatidylcholine: phosphatidylserine lipid monolayer. Using this surface the interaction of unlabelled FVIII in concentrates was observed from which direct kinetic data (kon, koff and KD values) were obtained in real-time. Marked differences in the binding to PL, as measured by KD values, between different products were observed. These fell into three categories: two recombinant FVIII products showed high affinities for PL with KD values around 0. 05-0.14 nM; four high-purity plasma derived products, two prepared by monoclonal antibody and two prepared by ion-exchange chromatography, had 6-8-fold lower affinities, and two intermediate-purity products had 34-60-fold lower affinities with KD values in the nM region. Measurements of kon and koff values for each product showed that the differences in the KD values expressed were primarily due to the differences in their respective kon values, although the recombinant products showed changes in the koff values. The study showed that the assessment of binding to PL by FVIII in concentrates was possible without prior purification and gave KD values in the range reported previously for other methods. The difference between the products requires further investigation but may be partly due to other proteins present, in particular the content and quality of von Willebrand factor which is known to affect PL binding of FVIII.     

Kinetics of the interaction between anti‐FVIII antibodies and FVIII from therapeutic concentrates,with and without von Willebrand factor,assessed by surface plasmon resonance

The presence of VWF in plasma‐derived FVIII (pdFVIII/VWF) products has been pointed out as a key difference with recombinant FVIII (rFVIII) products with regard to immunogenicity. A Surface Plasmon Resonance (SPR) study was designed to characterize in detail the interaction between anti‐FVIII (IgGs) from a severe haemophilia A patient, and FVIII from concentrates of different sources. Full‐length rFVIII (preincubated or not with purified VWF), B domain‐deleted (BDD)‐rFVIII and pdFVIII/VWF were analysed. To ensure reproducible conditions for accurate determination of kinetic constants, a capture‐based assay format was developed using protein G surfaces for specific and reversible coupling of endogenous anti‐FVIII antibodies. Concentration ranges (nm ) of FVIII products tested were 9–0.03 (rFVIII) and 6–0.024 (pdFVIII/VWF). The association with antibodies was monitored for 3–5 min, whereas dissociation of the complex was followed for 5–20–240 min. A strong interaction of rFVIII and BDD‐rFVIII with patient's IgG was detected with the K _D values in the low picomolar range (5.9 ± 3.0 and 12.7 ± 6.9 pm , respectively) and very slow dissociation rates, while pdFVIII/VWF showed only marginal binding signals. The VWF complexed rFVIII displayed reduced binding signals compared with uncomplexed rFVIII, but the K _D was still in the picomolar range (4.1 ± 1.9 pm ) indicating insufficient complex formation. rFVIII, alone or bound to exogenously added VWF, showed high affinity for anti‐FVIII IgGs from a severe haemophilia A patient whereas pdFVIII/VWF did not. These results are in agreement with those studies that point towards rFVIII concentrates to be more immunogenic than pdFVIII concentrates.     

Continuous infusion of FVIII and FIX concentrates: in vitro analysis of clinically relevant parameters.

A high purity factor VIII/von Willebrand Factor (FVIII/vWF) concentrate (IMMUNATE [STIM plus]) (n = 6 batches), and a high purity factor IX (FIX) concentrate (IMMUNINE [STIM plus]) (n = 7 batches), were assessed in vitro for their applicability to continuous infusion. Parameters pertinent to continuous infusion were investigated and included stability, sterility and, in the case of FIX, the generation of potentially thrombogenic components. Four stationary or transportable mini infusion pumps, equipped with polyethylene, polypropylene or polyvinylchloride plastic components were used. The concentrates were reconstituted without extra filling volume and perfused at 12.5 mL h-1 and 1 mL h-1; sampling was carried out at the start of the experiment and for up to 48 h. The FVIII procoagulant activity (FVIII:C) was assayed by amidolytic, 1-stage and 2-stage assays; vWF was examined for ristocetin cofactor activity, antigen and multimers. The FIX coagulation activity (FIX:C) was determined by a 1-stage coagulation assay; thrombogenicity potential was assessed in vivo (Wessler stasis model in rabbits) and in vitro (FIXa and nonactivated thromboplastin time). Reconstituted concentrate incubated under the same conditions served as a control. Both concentrates remained sterile throughout the testing period. The perfused and control samples remained stable, retaining over 95% of activity for FVIII:C and over 90% for FIX:C for up to 48 h. Intermittent decrease of FVIII:C or FIX:C was not observed, suggesting no adsorption of FVIII or FIX onto plastic surfaces during either short or long-term exposure. No thrombogenic components were detected in the high purity FIX concentrate. Thus, under the in vitro conditions used, FVIII/vWF and FIX were found to be suitable for administration by continuous infusion.     

Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens

Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.     

Type specificity and significance of different isotypes of serum antibodies to human papillomavirus capsids

Isotype-specific serum antibody responses against human papillomavirus (HPV) type 16 were evaluated by use of cross-sectional, prospective, and population-based seroepidemiologic studies. IgG1 and IgA were the most abundant isotypes. No sample contained IgG2, and <25 samples contained IgG3 or IgM. Total IgG, IgA, and IgG1 were HPV type specific and were associated with HPV-16 DNA (odds ratios [ORs], 5.4, 5.0, and 5.9, respectively; P<.001) but not with other HPV DNA (ORs, 1.2, 1.2, and 0.8, respectively; P value was not significant). Total IgG and IgG1 were strongly associated with number of lifetime sex partners (P<.001); IgA was only associated with number of recent sex partners and lifetime sex partners among younger women. Total IgG, IgG1, and IgA were associated with cervical intraepithelial neoplasia type III and also predicted risk of future cervical neoplasia. IgG and IgG1 appeared to mark lifetime cumulative exposure, whereas IgA may mark recent or ongoing infection.     

Should hemophilia treaters switch to albumin-free recombinant factor VIII concentrates

PURPOSE OF REVIEW: To discuss the advantages and disadvantages of the albumin-free recombinant factor VIII concentrates in the treatment of hemophilia A. RECENT FINDINGS: The third-generation recombinant factor VIII product Advate has been found to be safe and effective in treating bleeding associated with hemophilia A. SUMMARY: Multiple issues must be considered when selecting a factor VIII concentrate for patients with hemophilia A including efficacy, availability, risk of transmission of infectious agents, risk of inhibitor development and cost. Third-generation recombinant factor VIII concentrates have been shown to be safe and effective. A theoretical improvement in risk of infectious agent transmission has been achieved by production of the products without human or animal plasma proteins. Controversy exists, however, with regard to a higher risk of inhibitor development with recombinant products. The higher cost of Advate can also potentially play a role in product choice. Overall every patient and their family must be presented with the advantages and disadvantages of all factor VIII concentrates, and be allowed to make an informed decision about which product to use for treatment.     

Characterization of recombinant gp120 and gp160 from HIV-1: binding to monoclonal antibodies and soluble CD4

We compared four preparations of recombinant HIV-1 envelope glycoprotein: mammalian (Chinese hamster ovary cells) gp120 (Celltech); baculovirus gp120 from American Biotechnologies Inc. (ABT) and from MicroGeneSys (MGS); and baculovirus gp160 (Institute of Virology, Oxford, UK). Each envelope glycoprotein binds to a neutralizing monoclonal antibody (MAb) directed against the V3 loop, confirming the integrity of this type-specific neutralization epitope. MGS gp120 binds abnormally well to a MAb which recognizes an epitope preferentially exposed on denatured gp120. Consistent with this finding, MGS gp120 binds to soluble CD4 (sCD4) with an affinity 50-100-fold lower than that of Celltech gp120. The affinity of Celltech gp120 from sCD4 is 2.3 nM, indistinguishable from that of gp120 extracted from HIV-1 virions. Baculovirus gp120 (ABT) and gp160 also have a high affinity for sCD4. A significant proportion of anti-gp120 antibodies in HIV-positive human sera recognize epitopes that are dependent on the mammalian glycosylation pattern, and a human HIV-positive serum inhibits the binding of mammalian gp120 to sCD4 five- to 10-fold more potently than it inhibits baculovirus gp120 binding to sCD4.