Juan I Jorquera

CSL Behring, KPD, Pennsylvania, United States

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Publications (26)114.85 Total impact

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    ABSTRACT: Introduction Fetal bovine serum (FBS) is an animal product used as a medium supplement. The animal origin of FBS is a concern if cultured stem cells are to be utilized for human cell therapy. Therefore, a substitute for FBS is desirable. In this study, an industrial, xeno-free, pharmaceutical-grade supplement for cell culture (SCC) under development at Grifols was tested for growth of human mesenchymal stem cells (hMSCs), cell characterization, and differentiation capacity. Methods SCC is a freeze-dried product obtained through cold-ethanol fractionation of industrial human plasma pools from healthy donors. Bone marrow-derived hMSC cell lines were obtained from two commercial suppliers. Cell growth was evaluated by culturing hMSCs with commercial media or media supplemented with SCC or FBS. Cell viability and cell yield were assessed with an automated cell counter. Cell surface markers were studied by indirect immunofluorescence assay. Cells were cultured then differentiated into adipocytes, chondrocytes, osteoblasts, and neurons, as assessed by specific staining and microscopy observation. Results SCC supported the growth of commercial hMSCs. Starting from the same number of seeded cells in two consecutive passages of culture with medium supplemented with SCC, hMSC yield and cell population doubling time were equivalent to the values obtained with the commercial medium and was consistent among lots. The viability of hMSCs was higher than 90%, while maintaining the characteristic phenotype of undifferentiated hMSCs (positive for CD29, CD44, CD90, CD105, CD146, CD166 and Stro-1; negative for CD14 and CD19). Cultured hMSCs maintained the potential for differentiation into adipocytes, chondrocytes, osteoblasts, and neurons. Conclusions The tested human plasma-derived SCC sustains the adequate growth of hMSCs, while preserving their differentiation capacity. SCC can be a potential candidate for cell culture supplement in advanced cell therapies.
    Stem Cell Research & Therapy 03/2015; 6(28). DOI:10.1186/s13287-015-0016-2 · 4.63 Impact Factor
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    ABSTRACT: Background: A promising approach for treating Alzheimer's disease relies on the net efflux of the amyloid-β (Aβ) peptide from the brain to peripheral plasma, as a result of plasma Aβ clearance promoted by plasma removal and therapeutic albumin replacement. Objective: To assess the binding of therapeutic albumin (Albutein®, Grifols) to monomeric and aggregated Aβ according to methods previously tested on the interactions between Aβ and research-grade albumin. Methods: Albumin integrity and the interactions with albumin stabilizers (octanoic acid and N-Ac-Trp) were assessed through one-dimensional (1D) 1H-NMR and saturation transfer difference (STD) NMR spectra. The interactions between monomeric Aβ1-40 and albumin were probed by 2D 1H-15 N HSQC spectra of labeled Aβ1-40. The formation of cross-β structured Aβ1-42 assemblies was monitored by ThT fluorescence. The interactions between self-assembled Aβ1-42 and albumin were probed by Trp fluorescence. Results: NMR spectra indicated that both therapeutic and research-grade albumin are similarly well-folded proteins. No significant changes in either HSQC peak position or intensity were observed upon addition of albumin to 15N-labeled Aβ1-40, which rules out binding of albumin to monomeric Aβ with dissociation constant in the μM or lower range. When aggregated Aβ1-42 was added to albumin, quenching of Trp fluorescence was observed, which indicates albumin binding to Aβ1-42 aggregates. The relative potency of therapeutic albumin as an Aβ self-association inhibitor was in the same order of magnitude as research-grade albumin. Conclusions: Albutein® inhibited Aβ self-association by selectively binding Aβ aggregates rather than monomers and by preventing further growth of the Aβ assemblies.
    Journal of Alzheimer's disease: JAD 09/2013; 38(4). DOI:10.3233/JAD-131169 · 3.61 Impact Factor
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    ABSTRACT: Studies have demonstrated that traces of activated factor XI (FXIa) present in specific brands of intravenous immunoglobulin (IVIG) concentrates may pose a thrombogenic risk. To characterize procoagulant activity during fractionation and the elimination capacity of the Flebogamma(®) DIF (Grifols' IVIG) manufacturing process. Flebogamma(®) DIF fractionation steps included cryoprecipitate supernatant (Cryo/S), Fraction (Fr) I supernatant, and Fr II + III suspension. Purification steps included ultrafiltrate I, acid treatment, and pasteurization. Samples were assessed for total protein, IgG, and procoagulant activation markers. Cryo/S showed no procoagulant activity for prekallikrein activator (PKA), kallikrein-like, and non-activated partial thromboplastin time (NaPTT) with normal (-PPP) or FXI-deficient (-FXI) platelet poor plasma. Thrombin generation test (TGT)-PPP and TGT-FXI were <83-148 and <53-197 nM thrombin, respectively. Shortened NaPTTs (100-296 s), high PKA (51-119 IU/mL), kallikrein-like activities (0.043-0.075 ΔAU/min), positive TGTs (98-298 nM), and FXIa (9.5-14.0 ng/mL) were detected in Fr II + III. After pasteurization, no residual evidence of any procoagulant activity marker was observed, including the final IVIG concentrate at 5% or 10% protein. Results were similar in Fr II + III from different IVIG manufacturing facilities. The Flebogamma(®) DIF production process is capable of eliminating procoagulant activity because of its purification steps.
    Biologicals 09/2013; 41(6). DOI:10.1016/j.biologicals.2013.08.002 · 1.41 Impact Factor
  • Transfusion 05/2013; 53(5):1141-2. DOI:10.1111/trf.12140 · 3.57 Impact Factor
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    ABSTRACT: In this study, the virus-removal capacity of nanofiltration was assessed using validated laboratory scale models on a wide range of viruses (pseudorabies virus; human immunodeficiency virus; bovine viral diarrhea virus; West Nile virus; hepatitis A virus; murine encephalomyocarditis virus; and porcine parvovirus) with sizes from 18 nm to 200 nm and applying the different process conditions existing in a number of Grifols' plasma-derived manufacturing processes (thrombin, α1-proteinase inhibitor, Factor IX, antithrombin, plasmin, intravenous immunoglobulin, and fibrinogen). Spiking experiments (n = 133) were performed in process intermediate products, and removal was subsequently determined by infectivity titration. Reduction Factor (RF) was calculated by comparing the virus load before and after nanofiltration under each product purification condition. In all experiments, the RFs were close to or greater than 4 log10 (>99.99% of virus elimination). RF values were not significantly affected by the process conditions within the limits assayed (pH, ionic strength, temperature, filtration ratio, and protein concentration). The virus-removal capacity of nanofiltration correlated only with the size of the removed agent. In conclusion, nanofiltration, as used in the manufacturing of several Grifols' products, is consistent, robust, and not significantly affected by process conditions.
    Biologicals 01/2013; 42(2). DOI:10.1016/j.biologicals.2013.10.003 · 1.41 Impact Factor
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    ABSTRACT: BACKGROUND: The variant Creutzfeldt-Jakob disease incidence peaked a decade ago and has since declined. Based on epidemiologic evidence, the causative agent, pathogenic prion, has not constituted a tangible contamination threat to large-scale manufacturing of human plasma-derived proteins. Nonetheless, manufacturers have studied the prion removal capabilities of various manufacturing steps to better understand product safety. Collectively analyzing the results could reveal experimental reproducibility and detect trends and mechanisms driving prion removal. STUDY DESIGN AND METHODS: Plasma Protein Therapeutics Association member companies collected more than 200 prion removal studies on plasma protein manufacturing steps, including precipitation, adsorption, chromatography, and filtration, as well as combined steps. The studies used a range of model spiking agents and bench-scale process replicas. The results were grouped based on key manufacturing variables to identify factors impacting removal. The log reduction values of a group are presented for comparison. RESULTS: Overall prion removal capacities evaluated by independent groups were in good agreement. The removal capacity evaluated using biochemical assays was consistent with prion infectivity removal measured by animal bioassays. Similar reduction values were observed for a given step using various spiking agents, except highly purified prion protein in some circumstances. Comparison between combined and single-step studies revealed complementary or overlapping removal mechanisms. Steps with high removal capacities represent the conditions where the physiochemical differences between prions and therapeutic proteins are most significant. CONCLUSION: The results support the intrinsic ability of certain plasma protein manufacturing steps to remove prions in case of an unlikely contamination, providing a safeguard to products.
    Transfusion 12/2012; 53(9). DOI:10.1111/trf.12050 · 3.57 Impact Factor
  • Alzheimer's and Dementia 07/2012; 8(4):P410-P411. DOI:10.1016/j.jalz.2012.05.2045 · 17.47 Impact Factor
  • Montserrat Costa, Ana M Ortiz, Juan I Jorquera
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    ABSTRACT: Clearance of plasma amyloid-β (Aβ) through plasma exchange and replacement with therapeutic albumin to facilitate net Aβ efflux from the brain to plasma is a novel approach for the treatment of Alzheimer's disease. Therefore, thorough characterization of the capacity of therapeutic albumin to bind Aβ is warranted. In this study, Aβ40 and Aβ42 were quantified by commercial ELISA or Araclon ABtest® in samples of Grifols' therapeutic albumin (Albutein®) 5%, 20%, and 25%. The capacity of Albutein® to bind Aβ was assessed by: a) ELISA in serially diluted therapeutic albumin (0-45 mg/ml protein concentration) to which 80 pg/ml of synthetic Aβ peptide (sAβ40 or sAβ42) were added; b) ELISA in samples of the therapeutic albumin containing serially diluted sAβ40 or sAβ42 (60-400 pg/ml); and c) surface plasmon resonance (SPR) for sAβ42 binding. The Aβ content in Albutein® was below the quantification threshold of the ELISA tests (<25 to <62.5 pg/ml) and ABtest® (<3.125 pg/ml). Quantification of exogenously added sAβ42 decreased in parallel with increasing protein concentration (59-78% at 45 mg/ml albumin). Recovery of sAβ serially diluted in Albutein® was ∼60% for sAβ40 and ∼70% for sAβ42, but was ∼100% in control samples without albumin. The KD by SPR analysis for sAβ42 interaction with Albutein® was 1.72 ± 0.24 × 10-6 M. In conclusion, Grifols' therapeutic albumin has undetectable content of Aβ40 and Aβ42. Moreover, Grifols' therapeutic albumin consistently binds peptides containing the primary sequence of human Aβ.
    Journal of Alzheimer's disease: JAD 01/2012; 29(1):159-70. DOI:10.3233/JAD-2012-111139 · 3.61 Impact Factor
  • Montse Costa, Ana Maria Ortiz, Juan Ignacio Jorquera
    Alzheimer's and Dementia 07/2011; 7(4). DOI:10.1016/j.jalz.2011.05.353 · 17.47 Impact Factor
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    ABSTRACT: The variant Creutfeldt-Jakob disease (vCJD) is a transmissible spongiform encephalopathy (TSE) associated with the ingestion of cattle derived products affected with bovine spongiform encephalopathy. vCJD emerged in the UK, where most of the cases occurred (170 of 217 cases worldwide). Manufacturers of biological products must investigate the ability of their production processes to remove TSE agents. Two manufacturing steps (polyethylene glycol-PEG precipitation and nanofiltration down to 20 nm) of Flebogamma(®) DIF, were evaluated by western blot and bioassay to measure the prion protein (PrP(Sc)) and infectivity clearance capacity, respectively. A laboratory scale model representative of the industrial process and a (experimentally) spiked TSE model-agent (hamster scrapie strain 263 K) were employed. Both steps showed a significant capacity to clear the TSE model-agent used since no PrP(Sc) signal or infectivity was detected in the resulting product of each step. PEG precipitation and nanofiltration provided reduction factors of ≥6.19 log(10)ID(50) and ≥5.45 log(10)ID(50) respectively. Both steps showed consistency between western blot and bioassay results. These results demonstrate the ability of the Flebogamma(®) DIF manufacturing process to clear TSE agents beyond the limit of detection of the assays, by several orders of magnitude.
    Biologicals 11/2010; 38(6):670-4. DOI:10.1016/j.biologicals.2010.08.003 · 1.41 Impact Factor
  • Montserrat Costa, Ana María Ortiz, Juan Ignacio Jorquera
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    ABSTRACT: Most plasma beta-amyloid peptide (Alphabeta) has been described to circulate bound to albumin (approx. 90%). Moreover, a balance between peripheral and brain Alphabeta seems to exist, so a reduction of Alphabeta levels in blood through plasma exchange with therapeutic albumin should induce a clearance of brain Alphabeta. In this study, content of Alphabeta in therapeutic albumin as well as its binding capacity were characterized. Levels of Alphabeta(1-40) and Alphabeta(1-42) were determined by means of ELISA technique in therapeutic albumin (human albumin Grifols; n = 18 batches), in normal plasma (n = 8) and in plasma from patients with Alzheimer disease (n = 45). Binding capacity of therapeutic albumin to synthetic peptides containing the primary sequence of human Alphabeta(1-42) was determined by means of surface plasmon resonance (SPR) technique. RESULTS. Both the Alphabeta(1-40) and Alphabeta(1-42) levels in therapeutic albumin were always lower than the last valid point measured in the standard curve (< 25 to < 63 pg/mL). Levels in normal plasma and in plasma from Alzheimer disease patients ranged between < 25 to 312 pg/mL for Alphabeta(1-40), and < 25 to 279,4 pg/mL for Alphabeta(1-42). SPR studies confirmed the high affinity of therapeutic albumin for the experimental Alphabeta peptide. Human albumin Grifols shows undetectable Alphabeta levels, and lower to those observed in normal plasma and in plasma from patients with Alzheimer disease. Moreover, it was able to bind peptides containing the sequence of human Alphabeta(1-42).
    Revista de neurologia 03/2010; 50 Suppl 5:S1-4. · 0.93 Impact Factor
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    ABSTRACT: A new human liquid intravenous immunoglobulin product, Flebogamma DIF, has been developed. This IgG is purified from human plasma by cold ethanol fractionation, PEG precipitation and ion exchange chromatography. The manufacturing process includes three different specific pathogen clearance (inactivation/removal) steps: pasteurization, solvent/detergent treatment and Planova nanofiltration with a pore size of 20 nm. This study evaluates the pathogen clearance capacity of seven steps in the production process for a wide range of viruses through spiking experiments: the three specific steps mentioned above and also four more production steps. Infectivity of samples was measured using a Tissue Culture Infectious Dose assay (log(10) TCID(50)) or Plaque Forming Units assay (log(10) PFU). Validation studies demonstrated that each specific step cleared more than 4 log(10) for all viruses assayed. An overall viral clearance between > or =13.33 log(10) and > or =25.21 log(10), was achieved depending on the virus and the number of steps studied for each virus. It can be concluded that Flebogamma DIF has a very high viral safety profile.
    Biologicals 03/2010; 38(4):486-93. DOI:10.1016/j.biologicals.2010.02.008 · 1.41 Impact Factor
  • Juan I Jorquera
    International immunopharmacology 12/2009; 10(3):373-4; author reply 375-6. DOI:10.1016/j.intimp.2009.12.007 · 2.71 Impact Factor
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    ABSTRACT: Instituto Grifols has developed two different sterile and ready to use anti-hepatitis B (anti-HB) enriched immunoglobulin concentrates: Niuliva® is a 5% intravenous immunoglobulin solution with 250 IU/ml anti-HB potency, and Gamma anti-hepatitis B Grifols® (Igantibe®in some countries) is a 16% intramuscular immunoglobulin solution with 200 IU/ml anti-HB potency. The production process includes careful plasma donor selection, analysis to discard specific markers of relevant viral infections in the individual donations and plasma pools and, in order to further increase the safety margin, steps aimed at eliminating potential pathogenic agents (e.g., pasteurization). Characterisation studies from both products showed high IgG purity (more than 99%) and an IgG subclass distribution similar to normal plasma. Other potential accompanying proteins (eg: IgA, IgM, albumin, transferrin, etc) were undetectable or very low. Results from additional parameters (identification, total protein, molecular distribution, etc.) meet European Pharmacopoeia's requirements. The stability profile indicates that the products are stable between 2°C and 8°C for two years (Gamma anti-hepatitis B Grifols®) or three years (Niuliva®), maintaining the anti-HB potency.
    Digestive and Liver Disease Supplements 12/2009; 3(4):112-118. DOI:10.1016/S1594-5804(09)60038-3
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    J I Jorquera
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    ABSTRACT: Flebogamma 5% dual inactivation and filtration (DIF), a new 5% liquid intravenous immunoglobulin with a stability of 2 years when stored at temperatures between 2 and 30 degrees C, has been developed. This new product is the result of the accumulated experience provided by Flebogamma, with more than 30 million grams administered since 1992 in Europe and the United States, and the implementation of the latest technology to improve Flebogamma even more by increasing its viral safety margin further. In addition to the specific inactivation stage for Flebogamma 5% (pasteurization), the new process includes a solvent-detergent treatment and nanofiltration through a Planova filter down to 20 nm. The preparation presents a mean purity of 99.6 +/- 0.2% with a correct chromatographic profile. Percentage values of immunoglobulin (Ig)G subclasses are equivalent to the physiological values of normal serum. The content in IgA as well as other possible impurities is very low, and the product presents a mean result of 109 +/- 5% in the Fc fragment functionality assay, demonstrating the integrity of the IgG molecule. The functionality is also reflected in neutralization tests carried out against poliomyelitis, diphtheria, measles and vaccinia which, apart from the antibody titres determined by enzyme-linked immunosorbent assay, guarantees that antibodies are capable of reacting against these pathogens. Regarding safety, the combination of multiple methods with capacity to inactivate or remove biological agents which include chemical inactivation, heat inactivation, nanofiltration and precipitations, with very different mechanisms of action, provides Flebogamma 5% DIF very wide margins of safety regarding to potential pathogens.
    Clinical & Experimental Immunology 09/2009; 157 Suppl 1:17-21. DOI:10.1111/j.1365-2249.2009.03953.x · 3.28 Impact Factor
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    ABSTRACT: The variant Creutzfeldt-Jakob disease (vCJD) is a transmissible spongiform encephalopathy (TSE), mainly present in the UK and is associated with the ingestion of bovine products affected with bovine spongiform encephalopathy. Manufacturers of biological products must investigate the ability of their production processes to remove TSE agents. We studied the purification steps in the manufacturing process of two FVIII/VWF concentrates (Alphanate) and Fanhdi in their ability to eliminate an experimental TSE-model agent. Hamster scrapie strain 263K brain-derived materials were spiked into samples of the solutions taken before various stages during its production: 3.5% polyethylene glycol (PEG) precipitation, heparin affinity chromatography and saline precipitation/final filtrations. PEG precipitation and affinity chromatography were studied both as isolated and combined steps. TSE agent removal was determined using a laboratory scale model representative of the industrial manufacturing process. The prion protein (PrP(Sc)) was measured with Western blot and TSE infectivity was measured with bioassay. Western blot results were in agreement with those obtained by bioassay, showing a significant removal capacity in the production process: 3.21-3.43 log(10) for the PEG precipitation; about 3.45 log(10) for the affinity chromatography; and around 2.0 log(10) for the saline precipitation plus final filtrations. PEG precipitation and heparin affinity chromatography were demonstrated to be two complementary TSE-model agent removal mechanisms with total removal being the sum of the two. An overall reduction factor of around 8 log(10) can be deduced. The tests from the production process of FVIII/VWF complex concentrates have demonstrated their potential for eliminating TSE agents.
    Haemophilia 07/2009; 15(6):1249-57. DOI:10.1111/j.1365-2516.2009.02067.x · 2.47 Impact Factor
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    ABSTRACT: Solvent/detergent (S/D) treatment is an established virus inactivation technology that has been applied in the manufacture of medicinal products derived from human plasma for more than 20 years. Data on the inactivation of enveloped viruses by S/D treatment collected from seven Plasma Protein Therapeutics Association member companies demonstrate the robustness, reliability, and efficacy of this virus inactivation method. The results from 308 studies reflecting production conditions as well as technical variables significantly beyond the product release specification were evaluated for virus inactivation, comprising different combinations of solvent and detergent (tri(n-butyl) phosphate [TNBP]/Tween 80, TNBP/Triton X-100, TNBP/Na-cholate) and different products (Factor [F]VIII, F IX, and intravenous and intramuscular immunoglobulins). Neither product class, process temperature, protein concentration, nor pH value has a significant impact on virus inactivation. A variable that did appear to be critical was the concentration of solvent and detergent. The data presented here demonstrate the robustness of virus inactivation by S/D treatment for a broad spectrum of enveloped test viruses and process variables. Our data substantiate the fact that no transmission of viruses such as human immunodeficiency virus, hepatitis B virus, hepatitis C virus, or of other enveloped viruses was reported for licensed plasma derivatives since the introduction of S/D treatment.
    Transfusion 06/2009; 49(9):1931-43. DOI:10.1111/j.1537-2995.2009.02222.x · 3.57 Impact Factor
  • Montserrat Costa, Anna Ma Ortiz, Juan Ignacio Jorquera
  • J I Jorquera
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    ABSTRACT: Two main types of safety procedures must be applied to biological products, including plasma derivatives: (i) preventive procedures and (ii) elimination procedures. Prevention includes epidemiological control of donor populations; checks on each donor's health condition; analysis of each donation for the main pathogens using serological methods; additional analysis of all plasma for human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis A virus (HAV) and the B19 virus, using nucleic acid amplification techniques (NAT). A 60 days or longer inventory hold of all plasma donations is applied, to allow additional time to discard previous donations from potential seroconverting or otherwise rejectable donors. Elimination procedures minimize the low residual risk of transmitting pathogens, including unknown or previously undetected ones. Since the introduction 20 years ago of solvent-detergent treatment, very effective against enveloped viruses (HIV, HBV, HCV, West Nile virus, SARS, avian influenza virus etc), there have been no known cases of transmission of this type of pathogens by products manufactured according to this procedure. Other inactivation procedures such as pasteurization, dry-heat or nanofiltration may prove equally effective. In addition, dry-heat treatment and nanofiltration are capable of effectively eliminating non-enveloped viruses (HAV, B19 virus). Recent studies show that the B19 virus is much more sensitive to heat (in lyophilized state or by pasteurization) and acid pH than previously thought. Although there is no evidence for the transmission of classic transmissible spongiform encephalopathies (TSEs) through blood or blood-products transfusion, four possible cases have been reported in the United Kingdom involving transmission by non-leukoreduced blood components of the agent that causes variant Creutzfeldt-Jakob Disease (vCJD), a disease linked to the outbreak of bovine spongiform encephalopathy (BSE) which took place in that country. However, there are no cases of human TSE (classic or variant) transmission by plasma-derived products. Analytical methods capable of detecting the vCJD agent in patients' brains (where high titres are found) and other tissues (such as the spleen, appendix and lymph nodes, where much lower concentrations are found) are unable to detect the agent in blood or plasma from patients with vCJD, even in the clinical phase of the disease. Experiments by Grifols and other groups show that the capacity of the production processes to eliminate vCJD agent models is many orders of magnitude greater than the maximum expected load of the agent. In this regard, the efficacy of precipitation, affinity chromatography, depth filtration and nanofiltration are particularly notable.
    Haemophilia 01/2008; 13 Suppl 5:41-6. DOI:10.1111/j.1365-2516.2007.01578.x · 2.47 Impact Factor

Publication Stats

127 Citations
114.85 Total Impact Points


  • 2009
    • CSL Behring
      KPD, Pennsylvania, United States
  • 2003
    • Grifols
      Barcino, Catalonia, Spain