Evaluation of the hemostatic potential including thrombin generation of three different therapeutic pathogen-reduced plasmas.

BACKGROUND AND OBJECTIVES: Several pathogen inactivation methods currently applied to therapeutic plasma may result in products with different hemostatic properties. This study aims at evaluating and comparing the hemostatic potential of different therapeutic plasma preparations currently available in France. MATERIALS AND METHODS: We studied three types of pathogen-reduced plasma for transfusion (MB/light, Amotosalen/UVA, industrial S/D plasma). Quarantine, non-pathogen-reduced plasma, was used as a control. This study compared more specifically the content in FVIII, fibrinogen (clottable and antigen assays) and ADAMTS-13 and evaluated the intrinsic hemostatic properties using a thrombin generation test [Calibrated Automated Thrombogram (CAT)] at high and low concentrations of tissue factor to assess the maximum quantity of thrombin generated or the contribution of FVIII and FIX in the amplification phase of thrombin generation, respectively. RESULTS: The median FVIII concentration was >70 IU/dl for each preparation. Endogenous thrombin potential values were significantly different among the methods of plasma preparation (P<0·001) but were all in the range of the values measured in donors' plasma. Control by the thrombomodulin-activated protein C system was preserved in all preparations (>50% inhibition of endogenous thrombin potential). Fibrinogen concentrations were all within normal range but fibrinogen levels were lower in the plasmas treated with photochemical methods. ADAMTS-13 levels were preserved. CONCLUSION: The hemostatic potential appears well preserved in all therapeutic plasmas tested but there are some differences between preparations, the clinical relevance of which remains to be elucidated.

Isolation and characterization of a low molecular weight growth-promoting factor from human plasma.

A low molecular weight growth factor (LMW-GF) enriched preparation was purified from human plasma after ultrafiltration or gel filtration by means of molecular sieving chromatography low pressure reversed phase chromatography (LP-RPLC) and electrophoresis. Purification was monitored by a biological assay testing the capacity of the fractions to enhance the sulfation activity of the somatomedins/insulin-like growth factors on chick embryo cartilage. Analysis of its chemical nature show that it is hydrophilic, stable to heat, resistant to most of the proteases but that it is degraded by acid hydrolysis or carboxypeptidase Y action. UV absorption spectrum and ion-exchange chromatographic retention behavior support the hypothesis that the most purified active preparation includes a peptide structure. The presence of sugar is suggested by concanavalin A binding experiments. The fact that the purification fractions also enhance thymidine uptake by other cell lines (fibroblasts, activated lymphocytes) widens the role of such small plasma molecules in the field of growth factor activities.

[Validation of the virus inactivation capacity of a procedure of human plasma albumin purification by chromatography]. / Validation des capacités d'inactivation des virus d'un procédé de purification de l'albumine plasmatique humaine par chromatographie.

Almost the whole of the human plasma albumin preparations intended for clinical or biological uses is at present fractionated by cold ethanol precipitation technics based on the Cohn method. However, ion-exchange chromatographic processes have been recently developed. The aim of this work was the evaluation of the viral inactivation efficacy of an automated industrial chromatographic process allowing fractionation of 350 to 400 l of plasma per cycle (one precipitation step, three ion-exchange chromatography steps using the Spherodex-Spherosil gels--Sepracor-IBF, Villeneuve la Garenne, France--and one pasteurization step. Three relevant viruses were selected for this validation study: the hepatitis B virus (HBV), the poliomyelitis virus and the human immunodeficiency virus (HIV). In order to comply with EEC and FDA regulatory documents, significant amounts of the tested viruses were spiked into the different fractions obtained during the various purification steps and their removal or inactivation during the subsequent step were determined. The validation study was performed under conditions which mimic the manufacturing process using fractions obtained during a semi-industrial fractionation. Moreover, residual viral infectivity was checked on after elution and washing of the columns for each chromatographic step. Results have pointed out: a) an overall reduction of 4.4 log 10 for HBV. Infectivity is judged by a combination of several markers and the DNA polymerase activity is the most affected particularly during the three ending purification steps; b) an overall reduction in virus titer > 10 log 10 for the poliomyelitis virus; c) an overall reduction in virus titer > 10 log 10 for HIV (four of the five steps have an important potential to inactivate this virus increasing the safety of the process). Moreover, no residual viral infectivities were detected after washing of the columns. In conclusion, this study showed the viral safety of human albumin purified using the chromatographic Spherodex-Spherosil process. As had been observed for fractionation by means of ethanol, the pasteurization step is necessary to ensure inactivation of two of the three viruses tested (HBV and poliomyelitis virus). This validation study allowed the preparation of a manufacturing and controls document for albumin and a marketing authorization has been issued by the "Laboratoire National de la Santé" (LNS, France).

Does Tris-HCl effectively participate in transamination during hemoglobin pyridoxylation?

To test whether Tris is required for covalent binding of pyridoxal phosphate (PLP) to hemoglobin, we carried out the reaction in solutions of Tris homologues, carrying a blocked amine function. With the exception of Mono-Tris, these compounds permitted the synthesis of modified hemoglobins with acceptable spectral properties, P50 values, cooperativity and methemoglobin content, refuting Tris HCI participation during hemoglobin pyridoxylation.

[Human plasma fibronectin. Comparison of methods for preparation of a concentrate for therapeutic use from different sources]. / Fibronectine plasmatique humaine. Comparaison de méthodes de préparation d'un concentré à usage thérapeutique à partir de différentes sources.

Three methods, successive precipitations, affinity chromatography on immobilized gelatin and immunoaffinity chromatography with monoclonal anti-fibronectin antibodies were optimized and compared in order to be used for large scale preparation of human plasma fibronectin (Fn). The functional properties of the various Fn preparations were investigated by means of two assays: quantitation of the gelatin-binding activity by ELISA and quantitation of the Fn-mediated attachment of fibroblasts on plastic. Functional alterations of the purified Fn were observed when it was isolated by successive precipitations. Both chromatographic methods provide a rapid and convenient way for isolation of pure and functional Fn. Mass production of monoclonal antibodies is too expensive and legislative requirements for the therapeutic use of monoclonal antibodies are limiting factors for the choice of immunopurification as large scale isolation procedure. Plasma Fn can be isolated from different sources: fresh frozen plasma, cryoprecipitate supernatant or by-products from factor VIII preparation. When gelatin-Sepharose chromatography is performed under optimized conditions, fibronectins isolated from these sources show similar properties. Large scale purifications of Fn from a by-product of factor VIII preparation were performed either by gelatin affinity chromatography or by successive precipitations. These two purification methods can be easily scaled-up since the data obtained closely correlate with analytical results. The chromatographic method supplies a higher purified (98 vs 75%) and functional (95 vs 50%) material when compared with successive precipitations. Yield is also higher (50 vs 26%). The starting material undergoes viral inactivation and the affinity purified Fn, sterile, atoxic, apyrogen, which can be freeze-dried without additives fulfils all requirements for an injectable product.

Haemoglobin pyridoxalation in phosphate buffer: comparison of results with those obtained with Tris-HCl buffer.

Most workers studying haemoglobin solutions re-establish a P50 close to physiological values by covalent fixation of pyridoxal phosphate (PLP) using the method first described by Benesch. This is performed with deoxyhaemoglobin in a Tris-HCl buffer, considered to be necessary for transimination. To simplify the chemical procedure of the conjugation of a macromolecule to Hb-PLP we have performed the pyridoxalation in a phosphate buffer. Physico-chemical analysis of pyridoxalated haemoglobin solutions show a slight degradation of the protein, a amount of fixed PLP to the haemoglobin and a right-shift of the dissociation curve similar whether the coupling is performed in the phosphate or the Tris buffer. The pharmacological evaluation by total and isovolumic exchange transfusion in rats, of solutions prepared from both methods of pyridoxalation show identical survival times, vascular persistence and stability of the conjugates. Thus the pyridoxalation takes place with a comparable yield whatever reagent is used, proving that Tris is not essential to haemoglobin pyridoxalation.

Early rupture and degeneration of cryopreserved arterial allografts.

Arterial allografts are used in vascular surgery to solve a major problem: vascular reconstruction in the infected area. To palliate the unavailability and to reduce the risk of viral disease transmission, vascular allografts are currently cryopreserved and stored in tissue banks. In our recent clinical experience, we observed several cases of rupture and degeneration of cryopreserved arterial allografts. All indications are that current cryopreservation protocols are probably the cause for these degenerations.

Purification of biologically active human plasma transthyretin by dye-affinity chromatography: studies on dye leakage and possibility of heat treatment for virus inactivation.

The application of a purification procedure for the industrial preparation from human plasma of a therapeutic protein may be hindered by several safety concerns. The dye leaching from Remazol Yellow GGL-Sepharose used for the affinity chromatography of human plasma transthyretin was quantitatively studied by a sensitive competitive enzyme immunoassay. The possibility of including a heat treatment step for virus inactivation in the purification process while preserving the biochemical and functional characteristics of the protein is also reported.

Purification and characterization of three molecular forms of insulin-like growth factor II from human Cohn paste IV.

The somatomedins or insulin-like growth factors (IGFs) are a family of peptides present in human serum. They are bound to specific carrier proteins and are thought to mediate growth-promoting actions of human growth hormone. Starting from Cohn fraction IV of human plasma, we describe here a rapid and highly efficient procedure for the purification to homogeneity, in addition to IGF I. of three forms of insulin-like growth factor II: IGF IIA (10-12 kDa), IGF IIB (the "classical" 7.5 kDa IGF II) and IGF IIC, identified as the IGF II variant of Jansen by fast-atom bombardment mass spectrometry. The procedure is based on ion-exchange chromatography and gel permeation chromatography on Biogel P10. As judged by specific radioimmunoassay methods for IGF I and IGF II, one of the most striking advantages of this process at this stage is the yield of IGF I not contaminated by 7.5 kDa IGF II. Isoelectric focusing or chromatofocusing, which require affinity chromatography to separate proteins from the polybuffers, are not necessary in this procedure. Final purification was directly achieved by preparative, followed by analytical high-performance liquid chromatography. The N-terminal sequence of peptide IGF IIB (39 amino acids) and peptide IGF I (29 amino acids) showed total homology with those previously described by Rinderknecht and Humbel [FEBS Lett., 89 (1978) 283]. The final yields of purified human IGF I and IGF IIB were 15 and 25 micrograms, respectively, from 11 of serum. All peptides interact with specific receptors on human lymphocytes and red blood cells, and are biologically active (stimulation of 35S uptake, increasing [3H]thymidine incorporation in human and chick embryo fibroblasts).

The biological assay of human somatomedin A: improvement by small molecular weight natural molecules.

Somatomedin activity is often measured by 35SO4 uptake in pelvic chick embryo cartilage. This assay gives good results when the somatomedin activity is measured in serum, but often gives uninterpretable results for purified somatomedin. An ultrafiltrate (UF 1000) obtained from human normal serum, added to the incubation medium allows measurement of biological somatomedin activity even when somatomedin is highly purified. UF 1000 contains some compounds of molecular weight lower than 1000 daltons. UF 1000 acts either when used simultaneously with somatomedin or in pre-incubation with cartilage before testing biological activity. So, we chose to use it in pre-incubation (1 h) at the optimal concentration of 10% (v/v), the standard curves being realized with normal serum retentates . In these conditions, the proper action of UF 1000 does not interfere with calculation of somatomedin activity. This method has enabled us to show that a diminution of serum somatomedin activity can be linked either to an anomaly of UF 1000 or to a somatomedin deficiency.