J I Jorquera

CSL Behring, KPD, Pennsylvania, United States

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Publications (69)226.94 Total impact

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    José M Diez · Ewa Bauman · Rodrigo Gajardo · Juan I Jorquera
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    ABSTRACT: 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. 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. 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. 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.
    Full-text · Article · Mar 2015 · Stem Cell Research & Therapy
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    M. I. Bravo · B. Da Rocha‐Souto · S. Grancha · J. I. Jorquera
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    ABSTRACT: Sensitivity to FVIII inhibitors of the native plasma-derived (pd) FVIII/VWF complex vs. the complexes formed after exogenous FVIII infusion in the haemophilic patient has not been thoroughly studied. The role of VWF in the interaction of FVIII with inhibitors was studied in vitro using different combinations of VWF and FVIII concentrates. Normal plasma, pdFVIII/VWF and isolated FVIII (recombinant FVIII, B-domain deleted and pdFVIII) were used. Titre (BU) was kinetically determined (up to 2 h) in serial dilutions of inhibitor IgG (purified from a pool of plasmas with inhibitors) mixed with VWF and then incubated with the different FVIII. Inhibitor was also added to previously mixed VWF+FVIII. Residual FVIII:C was determined. TGA assays were performed with FVIII-deficient plasma spiked with the FVIII-VWF mixtures with/without an ESH-8 antibody. Inhibitor titres for plasma and pdFVIII/VWF were comparable at all time points. Titres for all concentrates of isolated FVIII were significantly higher than those for plasma or pdFVIII/VWF (1.4–1.9 fold) even after preincubation with VWF. At t = 0 h, titres for plasma or pdFVIII/VWF were unquantifiable, but were detectable for isolated FVIII (0.6–1.6 BU). In contrast to pdFVIII/VWF, the decrease in thrombin generation parameters by isolated FVIII in the presence of ESH-8 was significant (P < 0.01) even when previously combined with VWF. In conclusion, VWF protection against FVIII inhibitor activity might be higher with native pdFVIII/VWF complex than with the corresponding compound formed from the isolated proteins. Bethesda assay titration using different FVIII concentrates would be advisable to guide the treatment of inhibitor patients.
    Full-text · Article · Aug 2014 · Haemophilia
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    ABSTRACT: Introduction: There is a growing interest in new therapeutic strategies for the treatment of Alzheimer disease (AD) which focus on reducing the beta-amyloid peptide (Aβ) burden in the brain by sequestering plasma Aβ, a large proportion of which is bound to albumin and other proteins. This review discusses the concepts of interaction between Aβ and albumin that have given rise to AMBAR (Alzheimer's Disease Management by Albumin Replacement) project, a new multicentre, randomised, controlled clinical trial for the treatment of AD. Development: Results from preliminary research suggest that Albutein(®) (therapeutic albumin, Grifols) contains no quantifiable levels of Aβ. Studies also show that Albutein(®) has Aβ binding capacity. On the other hand, AD entails a high level of nitro-oxidative stress associated with fibrillar aggregates of Aβ that can induce albumin modification, thus affecting its biological functions. Results from the phase ii study confirm that using therapeutic apheresis to replace endogenous albumin with Albutein(®) 5% is feasible and safe in patients with AD. This process resulted in mobilisation of Aβ and cognitive improvement in treated patients. The AMBAR study will test combination therapy with therapeutic apheresis and haemopheresis with the possible leverage effect of Albutein(®) with intravenous immunoglobulin replacement (Flebogamma(®) DIF). Cognitive, functional, and behavioural changes in patients with mild to moderate AD will be assessed. Conclusions: the AMBAR study represents a new therapeutic perspective for AD.
    Full-text · Article · Jul 2014 · Neurologia (Barcelona, Spain)
  • M. Bravo · B. Da Rocha-Souto · S. Grancha · J. Jorquera

    No preview · Article · Jun 2014 · Journal of Thrombosis and Haemostasis
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    ABSTRACT: Introduction There is a growing interest in new therapeutic strategies for the treatment of Alzheimer disease (AD) which focus on reducing the beta-amyloid peptide (Aβ) burden in the brain by sequestering plasma Aβ, a large proportion of which is bound to albumin and other proteins. This review discusses the concepts of interaction between Aβ and albumin that have given rise to AMBAR (Alzheimer's Disease Management by Albumin Replacement) project, a new multicentre, randomised, controlled clinical trial for the treatment of AD. Development Results from preliminary research suggest that Albutein® (therapeutic albumin, Grifols) contains no quantifiable levels of Aβ. Studies also show that Albutein® has Aβ binding capacity. On the other hand, AD entails a high level of nitro-oxidative stress associated with fibrillar aggregates of Aβ that can induce albumin modification, thus affecting its biological functions. Results from the phase ii study confirm that using therapeutic apheresis to replace endogenous albumin with Albutein® 5% is feasible and safe in patients with AD. This process resulted in mobilisation of Aβ and cognitive improvement in treated patients. The AMBAR study will test combination therapy with therapeutic apheresis and haemopheresis with the possible leverage effect of Albutein® with intravenous immunoglobulin replacement (Flebogamma® DIF). Cognitive, functional, and behavioural changes in patients with mild to moderate AD will be assessed. Conclusions the AMBAR study represents a new therapeutic perspective for AD.
    No preview · Article · Jan 2014
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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.
    No preview · Article · Sep 2013 · Journal of Alzheimer's disease: JAD
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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.
    No preview · Article · Sep 2013 · Biologicals
  • Montserrat Costa · Ana Maria Ortiz · Marylin Rosa-Bray · Juan Ignacio Jorquera

    No preview · Article · Jul 2013
  • Rodrigo Gajardo · Nathan J Roth · Douglas C Lee · Juan I Jorquera

    No preview · Article · May 2013 · Transfusion
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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.
    No preview · Article · Jan 2013 · Biologicals
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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.
    No preview · Article · Dec 2012 · Transfusion
  • Salvador Grancha · Steven Herring · Antonio Paéz · Pere Ristol · Juan Ignacio Jorquera
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    ABSTRACT: This chapter contains sections titled: Biochemistry and Physiology Production Processes for Factor IX Concentrates High-Purity Factor IX Pharmacopoeial Monographs Clinical Aspects High-Purity Factor IX and Self-Sufficiency References
    No preview · Chapter · Dec 2012

  • No preview · Article · Jul 2012 · Alzheimer's and Dementia
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    ABSTRACT: 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.
    Full-text · Article · May 2012 · Haemophilia
  • 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.
    No preview · Article · Jan 2012 · Journal of Alzheimer's disease: JAD
  • Montse Costa · Ana Maria Ortiz · Juan Ignacio Jorquera

    No preview · Article · Jul 2011 · Alzheimer's and Dementia
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    Nuria Marzo · Marta Jose · Laura Lopez · Mariona Bono · Maite Lopez · Juan I Jorquera

    Preview · Article · May 2011
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    S Grancha · R Navajas · C Marañón · A Paradela · J P Albar · J I Jorquera

    Full-text · Article · Feb 2011 · Haemophilia
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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.
    No preview · Article · Nov 2010 · Biologicals
  • 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).
    No preview · Article · Mar 2010 · Revista de neurologia

Publication Stats

529 Citations
226.94 Total Impact Points

Institutions

  • 2009
    • CSL Behring
      KPD, Pennsylvania, United States
  • 2003
    • Grifols
      Barcino, Catalonia, Spain
  • 1985-1995
    • Hospital Universitari i Politècnic la Fe
      • Departamento de Biopatología Clínica
      Valenza, Valencia, Spain