CAF-DCF Product Development Division
Contact person: Ruth Laub PhD
The goal of the CAF-DCF Product Development Department is to ensure both the pre-clinical and clinical efficacy of plasma-derived medicinal products and their biological safety regarding pathogens, pollutants, and accompanying proteins. Focusing on therapeutic proteins (IVIGs, albumin, fibrinogen, AGP, FVIII) in starting plasma pools and final concentrates, the Department has developed both immunological methods and biochemical-biophysical techniques, including predictive epitope-mapping algorithms for FVIII proteins and B19 NS1 and capsid proteins. Results are exploited in industrial applications (20 patents granted in the EU and US, 3 in 2011).
- Infection protective antibodies and sero-epidemiology
- Pneumococcal Ig in IVIG-treated infants, in relation to the standard for vaccines
- (Pro-)coagulant activities in intermediate Cohn fractions and IVIGs
- Albumin formulated with caprylate with or without N-Ac-tryptophan in a liver support device (Mars)
- B19 proliferation in hepatoblastoma cells
- Epidemiology of viral markers in donors in Belgium
- A new FVIII/vWF concentrate with 3 virus inactivation steps
- Key publications and patents
IVIGs, infection protective antibodies, and sero-epidemiology
Antibody-based therapy could be developed against most existing pathogens. Defining a general IgG level below which immunoglobulin should be given to primary or secondary immunodeficiency patients does not provide the main criterion for treatment. More relevant is the specific anti-infective protection restored after intravenous immunoglobulin (IVIG) administration. ELISAs are the most adequate techniques for measuring surrogates of protection against pathogens.
Capsular pneumococcal polysaccharides (PnPS) are the most recognized virulence factors of these organisms. Serotype-specific antibodies against PnPS provide protection against the corresponding serotypes. Today, about 30 serotypes are responsible for infections ranging from upper respiratory disease and otitis media to pneumonia, bacteraemia, and meningitis. Using robotics, liquid handling devices, and data processing, high-throughput quantification (HTQ) can manage the large number of serotypes and diversity of samples: plasma pools, IVIGs and patient samples collected in clinical studies. PnPS antibodies specific to 16 relevant pathogenic serotypes were determined by means of an ICH-Q2(R1)-validated HTQ process in the IVIG batches produced, showing very little variation over 7 years of production. A good correlation was found between PnPSAbs and the sero-epidemiology of the region where donors are living. Moreover, specific antibodies against Mycoplasma, Influenza A, Influenza B, Chlamydia, Rubella, Herpes Zoster and Herpes were quantified in plasma pools and IVIGs, demonstrating also the low variability of their levels in the different batches produced in different years.
In collaboration with D. Tuerlinkx1, B. Florkin2, I. De Schutter3, C. Chantrain4, F. Haerynck5, P. Philippet6 and A. Ferster7
Streptococcus pneumoniae remains a leading global cause of morbidity and mortality. For a vaccine, an anti-pneumococcal antibody level of 0.20 µg/ml is considered protective against invasive pneumococcal infection (WHO14 recommendations, 22F preabsorption). Patients with primary immunodeficiency disease (PID) are treated with immunoglobulin administered intravenously every 3-4 weeks and the PnPsAb levels required to minimize infection risk are not yet established. Because PnPsAb levels correlate with the infection risk, we have measured 16 major PnPsAbs just before (trough) and 1 hour after (peak) treatment with IVIG (Multigam®) in 22 pediatric PID patients over a period comprising 6 consecutive infusions in the context of a GCP open label multi-centre clinical study. The trough median percentage of measurements >0.2µg was 100% for all of the most prevalent serotypes tested (1, 3, 5, 6B, 7F, 8, 9N, 10A, 14, 18C, 19A, 19F, 23F) but not for the rare 12F (14%), 4 (7%), and 9V (92%). Pediatric PID patients treated with Multigam® have protective trough levels of PnPSAbs against the most prevalent pneumococcal serotypes.
In network with J. Bloem8, H. ter Hart8 and A. Koenderman8
In IVIG preparations, contact-activated contaminants with coagulant properties have been highlighted, with the recent voluntary withdrawal of Octagam® in the US and with suspension of its marketing authorization by the EMA. Following the EDQM resolution of June 2011 (monograph 0918), we developed and validated amydolytic assays targeting kallikrein, clotting assays for serine protease zymogens and active serine protease, and a thrombin generation assay to determine FXIa activity specifically. Activity was found in the intermediate Cohn fractions (I II III and I III) and was lower in Cohn fraction II. To identify the enzymes, accompanying proteins were separated from Ig molecules, concentrated, and analyzed by western blotting. FXII FXIIa, , FXI/FXIa, prekallikrein/kallikrein, and plasmin were found in intermediate plasma fractions but not in the final IVIG products (Mutigam® and Nanogam®) having undergone further purification steps.
In collaboration with T. De Bruyn9, B. Meijers10, P. Evenepoel10, L. Willems11, P. Augustijns9 and P. Annaert9
Mounting evidence suggests beneficial effects of albumin-dialysis-based liver support in patients suffering from acute-on-chronic liver failure. The molecular absorbent re-circulating system (MARS) is a non-biological liver support device based on the exchange of albumin-bound toxins between the patient's blood and a 20% human albumin solution in a secondary circuit. Bound toxins are removed by exposure to activated charcoal and a ion-exchange resin. The results showed a negative influence of the presence of N-Ac-tryptophan on the stability and binding capacity, particularly when warfarin was used as ligand. Effects on binding affinity were also observed. N-Ac-tryptophan (present in Baxter and Sigma albumins) was found to decrease the albumin binding affinity, as measured after 1-7 h of exposure to charcoal or resin. For albumin from CAF-DCF, which contains caprylate but no N-Ac-tryptophan, the binding affinity and capacity for warfarin were constant under all conditions tested.
In collaboration with L. Kubler12, ML Draps12 and A. Op De Beeck12
A new model for B19 proliferation was developed in hepatoblastoma cells. Multiplication was measured by Q-PCR. From only a few parvovirus B19 particles (100 IU), the cells were able to produce 3.107 IU B19-DNA over a 48-h culture period. Recently, FACS combined with B19 capsid immunodetection and microscopic immunofluorescence revealed that only a few cells are susceptible to be infected and give rise to a B19 progeny. Surprisingly, a high concentration of von Willebrand Factor (vWF) inhibits entry of B19 into susceptible cells. This may be due to the presence of the integrin ligand on vWF molecules capable of binding cell surface receptors.
The new guideline EMA/CPMP/BWP/548524/2008 provides additional guidance on reporting critical epidemiological data and estimating residual risk. Using the new calculation, the results show that the trends are not modified and that the HBV risk has been decreased by implementation of NAT on minipools of maximum 8 donation samples in the Blood Transfusion Centers.
In collaboration with GJ Derksen8, J. Penninks8 and A. Koenderman8
A new therapeutic FVIII concentrate rich in vWF was developed. The high vWF content presents several advantages: vWF exerts an immunoprotective effect on FVIII by reducing its immunogenicity and increasing its half-life. This therapeutic concentrate could also be used to treat several types of vWF diseases.
- Cliniques Universitaires U.C.L. de Mont-Godinne, Mont-Godinne, Belgium
- CHR La Citadelle, Liège, Belgium
- UZ Brussel, Brussels, Belgium
- UCL Saint-Luc, Brussels, Belgium
- UZ Gent, Gent, Belgium
- CHC Espérance, Liège, Belgium
- HUDERF, Brussels, Belgium
- Sanquin Plasma Products, Product Development, Amsterdam, The Netherlands
- KUL, Laboratory for Pharmacotechnology & Biopharmacy, Department of Pharmaceutical Sciences, Leuven, Belgium
- University Hospital Leuven, Division of Nephrology, Faculty of Medicine, Leuven, Belgium
- Univestity Hospital Gasthuisberg, Hospital Pharmacy, Department of Pharmaceutical Sciences, Leuven, Belgium
- ULB, Medecine Faculty, Epigénétique du Cancer, Brussels, Belgium
- Laub R, Strengers P. Intensive plasmapheresis donations contain less immunoglobulin and albumin: consequences for donor health. Vox Sang 2011;100(4):439.
- De Bruyn T, Meijers B, Evenepoel P, Laub R, Willems L, Augustijns P, Annaert P. Stability of therapeutic albumin solutions used for molecular adsorbent recirculating system-based liver dialysis. Artif Organs 2012; 36(1):29-41.
- Laub R, De Wael L. EP 0 771 324 B2 entitled "Concentré de fibrinogène issu de plasma sanguin, procédé et installation pour sa préparation" (granted February 2011).
- Laub R, Di Giambattista M. CA 2446390 entitled "Antigenic epitopes of factor VIII, inhibitors directed against said epitopes and use thereof" (granted June 2011).
- Laub R, Di Giambattista M. US 7,981,865 B2 entitled "Antigenic epitopes of factor VIII, inhibitors directed against said epitopes and use thereof" (granted July 2011).
- Laub R, Di Giambattista M. EP 1399556 B1 entitled "Antigenic epitopes of factor VIII, inhibitors directed against said epitopes and use thereof" (granted September 2011).