Removal and inactivation of human immunodeficiency virus (HIV-1) by cold ethanol fractionation and pasteurization during the manufacturing of albumin and immunoglobulins from human plasma

Viral safety is a prerequisite for manufacturing clinical albumin and immunoglobulins from human plasma pools. This study was designed to evaluate the efficacy of cold ethanol fractionation and pasteurization (60°C heat treatment for 10 h) for the removal inactivation of human immunodeficiency virus type 1 (HIV-1) during the manufacturing of albumin and immunoglobulins. Samples from the relevant stages of the production process were spiked with HIV-1, and the amount of virus in each fraction was quantified by the 50% tissue culture infectious dose (TCID₅₀). Both fraction IV fractionation and pasteurization steps during albumin processing were robust and effective in inactivating HIV-1, titers of which were reduced from an initial 8.5 log₁₀ TCID₅₀ to undetectable levels. The log reduction factors achieved were ≥4.5 and ≥6.5, respectively. In addition, fraction III fractionation and pasteurization during immunoglobulins processing were robust and effective in eliminating HIV-1. HIV-1 titers were reduced from an initial 7.3 log₁₀ TCID₅₀ to undetectable levels. The log reduction factors achieved in this case were ≥4.9 and ≥5.3, respectively. These results indicate that the process investigated for the production of albumin and immunoglobulins have sufficient HIV-1 reducing capacity to achieve a high margin of safety.     

Purification of IgG and albumin from human plasma by aqueous two phase system fractionation

The current shortages in human plasma products at global levels justify the development of new, cost effective plasma fractionation methods. We have developed a fractionation process to obtain immunoglobulin G (IgG) and albumin‐enriched fractions based on polymer‐salt aqueous two phase system (ATPS). A small‐scale (0.02 L) ATPS composed of polyethyleneglycol 3350 (PEG), potassium phosphate and sodium chloride, at pH 6.1, was evaluated and subjected to 50‐fold scale‐up (1 L). Further purification of the fractions was performed using caprylic acid precipitation and ion exchange chromatography. Similar yield and purity were obtained at both small and large scales. IgG precipitated in the PEG rich upper phase at 83% recovery and 2.75‐fold purification factor. An 81% pure albumin fraction was obtained in the salt rich bottom phase with a 91% yield. After polishing, IgG was obtained at a recovery of 70% and a purity of 92%. Corresponding values for albumin were 91% and 90%. This IgG and albumin fractionation technology deserves further evaluation as it may represent a potential alternative to conventional plasma fractionation methods. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1005–1011, 2012     

Polyacrylic acid based plasma fractionation for the production of albumin and IgG: Compatibility with existing commercial downstream processes

Commercial fractionation of human plasma into immunoglobulin- and albumin-rich fractions is often initiated with sequential cold ethanol-based precipitation methods, which have changed little over the past 70 years. The required low temperature (−4 to −8°C) and high concentrations of ethanol 8–40%) necessitate large-scale fixed processing lines, and major capital investment and operating costs. The resulting fractions are then further purified by ethanol based precipitation or chromatographic procedures to obtain the purified final product. Aqueous polyacrylic acid (PAA) based precipitation, which readily interfaces with existing downstream processing, could offer advantages with respect to cost, safety, environmental impact, and flexibility. Sequential precipitation with 7%, 12%, and 20% (w/v) solutions of PAA 8000 in the presence of a kosmotropic salt (sodium citrate) gave fibrinogen-, immunoglobulin-, and albumin-rich fractions with 80–90% yield and 64%, 55%, and 82% purity, respectively. Further purification of the IgG-rich precipitate by caprylic acid precipitation and anion exchange chromatography, achieved a target purity of >99%. This was also achieved for the downstream processing of the albumin-rich precipitate using a two-step ion exchange chromatographic procedure. This work shows that PAA precipitation can be used in place of cold ethanol precipitation to generate crude IgG and albumin fractions which can be purified to final products of acceptable purity.     

Removal and inactivation of hepatitis A virus during manufacture of urokinase from human urine

The purpose of the present study was to examine the efficacy and mechanism of the PAB (para-amino benzamidine) affinity column chromatography, Viresolve NFP virus filtration, pasteurization (60°C heat treatment for 10 h), and lyophilization steps employed in the manufacture of urokinase from human urine as regards the removal and/or inactivation of the hepatitis A virus (HAV). Samples from the relevant stages of the production process were spiked with HAV and subjected to scale-down processes mimicking the manufacture of urokinase. Samples were collected at each step, immediately titrated using a 50% tissue culture infectious dose (TCID₅₀), and the virus reduction factors evaluated. PAB chromatography was found to be an effective step for removing HAV with a log reduction factor of 3.24. HAV infectivity was rarely detected in the urokinase fraction, while most of the HAV infectivity was recovered in the unbound and wash fractions. HAV was completely removed during the vire solve NFP filtration with a log reduction factor of ≥4.60. Pasteurization was also found to be an effective step in inactivating HAV, where the titers were reduced from an initial titer of 7.18 log₁₀ TCID₅₀ to undetectable levels within 10 h of treatment. The log reduction factor achieved during pasteurization was ≥4.76. Lyophilization revealed the lowest efficacy for inactivating HAV with a log reduction factor of 1.48. The cumulative log reduction factor was ≥14.08. Accordingly, these results indicate that the production process for urokinase exhibited a sufficient HAV reducing capacity to achieve a high margin of virus safety.     

疏水层析结合冷乙醇沉淀纯化人血清白蛋白

将层析技术与冷乙醇工艺相结合用于人血清白蛋白的纯化 ,对各过程所采用的层析介质及层析条件进行了探索 ,得到了一条从人血浆中制备血清白蛋白的新路线 :将一步冷乙醇沉淀后的血浆上清进行脱盐除乙醇 ,用阳离子交换介质CMSepharoseFF以透过式层析的模式吸附非白蛋白组分 ,最后选用ButylSepharoseFF一步疏水层析后所得样品经SDS-PAGE银染显示一条单带 ,分析其纯度大于 99% ,计算工艺收率为 81.2%。与传统冷乙醇工艺相比较 ,该工艺最终样品纯度更高 ,且层析可以在常温下操作 ,易实现自动化控制.     

Removal and inactivation of viruses during the manufacture of a high-purity antihemophilic factor IX from human plasma

The purpose of this study was to evaluate the efficacy and mechanisms of the solvent/detergent (S/D) treatment, DEAE-toyopearl 650M anion-exchange column chromatography, heparin-sepharose 6FF affinity column chromatography, and Viresolve NFP filtration steps employed in the manufacture of high-purity antihemophilic factor IX (Green-Nine VF) from human plasma, with regard to removal and/or inactivation of blood-borne viruses. A variety of experimental model viruses for human pathogenic viruses, including human immunodeficiency virus (HIV), bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), hepatitis A virus (HAV), murine encephalomyocarditis virus (EMCV), and porcine parvovirus (PPV), were all selected for this study. Samples from relevant stages of the production process were spiked with each virus and subjected to scale-down processes mimicking the manufacture of high-purity factor IX. Samples were collected at each step, immediately titrated using a 50% tissue culture infectious dose (TCID₅₀), and virus reduction factors were evaluated. S/D treatment using the organic solvent, tri (n-butyl) phosphate (TNBP), and the detergent, Tween 80, was a robust and effective step in inactivation of enveloped viruses. Titers of HIV, BHV, and BVDV were reduced from the initial titer of 6.06, 7.72, and 6.92 log₁₀ TCID₅₀, respectively, reaching undetectable levels within 1 min of S/D treatment. DEAE-toyopearl 650M anion-exchange column chromatography was found to be a moderately effective step in the removal of HAV, EMCV, and PPV with log reduction factors of 1.12, 2.67, and 1.38, respectively. Heparin-sepharose 6FF affinity column chromatography was also moderately effective for partitioning BHV, BVDV, HAV, EMCV, and PPV with log reduction factors of 1.55, 1.35, 1.08, 1.19, and 1.61, respectively. The Viresolve NFP filtration step was a robust and effective step in removing all viruses tested, since HIV, BHV, BVDV, HAV, EMCV, and PPV were completely removed during the filtration step with log reduction factors of ≥ 5.51, ≥ 5.76, ≥ 5.18, ≥ 5.34, ≥ 6.13, and ≥ 4.28, respectively. Cumulative log reduction factors of HIV, BHV, BVDV, HAV, EMCV, and PPV were ≥ 10.52, ≥ 12.07, ≥ 10.49, ≥ 7.54, ≥ 9.99, and ≥ 7.24, respectively. These results indicate that the production process for GreenNine VF has a sufficient virus reduction capacity for achievement of a high margin of virus safety.     

Chronic and acute ethanol treatment modifies fluidity and composition in plasma membranes of a human hepaic cell line (WRL-68)

The aim of this study was to compare the effects of chronic (0.1 mol/L ethanol exposure during 30 days) and acute (0.5 mol/L ethanol exposure during 24 h) ethanol treatment on the physical properties and the lipid composition of plasma membranes of the WRL-68 cells (fetal human hepatic cell line). Using fluorescence polarization we found that ethanol treatment reduced membrane anisotropy due to disorganization of acyl chains in plasma membranes and consequently increased fluidity, as measured with the diphenylhexatriene probe. Addition of ethanolin vitro reduced anisotropy in control plasma membranes, whereas chronically ethanol-treated plasma membranes were relatively tolerant to thein vitro addition of ethanol. Acutely ethanol-treated plasma membranes exhibited a smaller anisotropy parameter value than control plasma membranes. We found a decrease in total phospholipid content in acute ethanol WRL-68 plasma membranes. Cholesterol content was increased in both ethanol treatments, and we also found a significant decrease in phosphatidylinositol and phosphatidylcholine and an increase in phosphatidylethanolamine content in ethanol-treated plasma membranes. Our data showed that ethanol treatment decreased the anisotropy parameter consistently with increased fluidity, while increasing the cholesterol/phospholipid ratio of plasma membranes of WRL-68 cells, but only chronically ethanol-treated plasma membranes exhibited tolerance to thein vitro addition of ethanol. It is important to note that some changes that were interpreted as a result of chronic ethanol treatment were also present in short-period ethanol treatments.Abbreviations DPH diphenylhexatriene - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SPH sphingomyelin     

Inactivation and removal of influenza A virus H1N1 during the manufacture of plasma derivatives

Although transmission of pandemic influenza A virus H1N1 2009 is still occurring globally, little has been reported about how this outbreak has affected the safety of plasma derivatives. To evaluate the safety of plasma derivatives, dedicated virus clearance processes used during their production were investigated for their effectiveness in eliminating this virus of recent concern. In this study, influenza A virus H1N1 strain A/NWS/33 (H1N1) was chosen as a surrogate. H1N1 was completely inactivated by fraction IV fractionation as well as pasteurization during the manufacture of albumin. H1N1 was also effectively removed into the precipitate by fraction III fractionation and completely inactivated by low pH incubation as well as pasteurization during the manufacture of intravenous immunoglobulin. H1N1 was completely inactivated within 1 min of solvent/detergent treatment using 0.3% tri (n-butyl) phosphate and 1.0% Triton X-100 and also completely inactivated within 10 min of dry-heat treatment at 98 °C during the manufacture of factor VIII. H1N1 was completely removed by virus filtration process using Viresolve NFP filter and also completely inactivated by pasteurization during the manufacture of anti-thrombin III. These results indicate that all the virus clearance processes commonly used have sufficient H1N1 reducing capacity to achieve a high margin of safety.     

Enhanced ethanol production,volatile compound biosynthesis and fungicide removal during growth of a newly isolated Saccharomyces cerevisiae strain on enriched pasteurized grape musts

The kinetic behavior of a newly isolated Saccharomyces cerevisiae strain, grown on pasteurized grape musts enriched with industrial sugars, was studied after the addition of various concentrations [0.0 (reference), 0.4 and 2.4 mg/L] of the fungicide quinoxyfen to the medium. Batch‐flask cultures were carried out. Significant quantities of biomass (10.0±0.8 g/L) were produced regardless of quinoxyfen addition to the medium; therefore, the addition of the fungicide did not seriously inhibit biomass production. Ethanol was synthesized in very high quantities in all trials (highest concentrations 106.4–119.2 g/L). A slight decrease of ethanol production in terms of both absolute value and conversion yield of ethanol produced per sugar consumed was, however, observed when the quinoxyfen concentration was increased. The addition of quinoxyfen led to significantly lower ethylic ester levels, which also pertains to the acetates analyzed in this study. Fusel alcohol synthesis seemed to be activated when 0.4 mg/L quinoxyfen was added, but at 2.4 mg/L of added fungicide, no statistically significant differences were observed compared with the control trial. Volatile acid levels did not present a uniform trend in relation with the added fungicide. Finally, the fermentation was accompanied by a significant reduction of the fungicide concentration (79–82 wt% fungicide removal).     

Combination mode of antimalarial drug mefloquine and human serum albumin: Insights from spectroscopic and docking approaches

The interaction between mefloquine (MEF), the antimalarial drug, and human serum albumin (HSA), the main carrier protein in blood circulation, was explored using fluorescence, absorption, and circular dichroism spectroscopic techniques. Quenching of HSA fluorescence with MEF was characterized as static quenching and thus confirmed the complex formation between MEF and HSA. Association constant values for MEF-HSA interaction were found to fall within the range of 3.79-5.73 × 10⁴ M^˗1 at various temperatures (288, 298, and 308 K), which revealed moderate binding affinity. Hydrogen bonds and hydrophobic interactions were predicted to connect MEF and HSA together in the MEF-HSA complex, as deduced from the thermodynamic data (ΔS = +133.52 J mol⁻¹ K⁻¹ and ΔH = +13.09 kJ mol⁻¹) of the binding reaction and molecular docking analysis. Three-dimensional fluorescence spectral analysis pointed out alterations in the microenvironment around aromatic amino acid (tryptophan and tyrosine) residues of HSA consequent to the addition of MEF. Circular dichroic spectra of HSA in the wavelength ranges of 200-250 and 250-300 nm hinted smaller changes in the protein's secondary and tertiary structures, respectively, induced by MEF binding. Noncovalent conjugation of MEF to HSA bettered protein thermostability. Site marker competitive drug displacement results suggested HSA Sudlow's site I as the MEF binding site, which was also supported by molecular docking analysis.     

Cucurbitacins from Ecballium elaterium juice increase the binding of bilirubin and ibuprofen to albumin in human plasma

Ecballium elaterium, a medicinal plant, whose fruit juice is used for the treatment of jaundice in folk medicine, has been reported as being capable of decreasing bilirubinemia in animals with jaundice [H.H. Elayan, M.N. Garaibeh, S.M. Zmeili, S.A. Salhab, Effects of Ecballium elaterium juice on serum bilirubin concentration in male rats, Int. J. Crude Drug Res. 27 (1989) 227-234]. The aim of this study is to identify the Ecballium elaterium components, which are able to modify the binding of bilirubin to albumin. The juice is fiber-free but contains proteins, lipids, sugars, and minerals. The extract of the juice, analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), contains cucurbitacins (Cuc) B, D, E, and I as well as several glycosylated compounds. Human plasma containing no or serial concentrations of Ecballium elaterium components were prepared and the direct bilirubin (DB) and total bilirubin (TB) were determined by the Jendrassik and Grof method. Our results showed that Cuc D, E, and B decreased the levels of DB and TB in plasma, while Cuc I, glycosyl derivatives, and proteins of the juice did not modify the bilirubin levels. The binding of domain specific ligands to HSA, bilirubin (domain IIA), and ibuprofen (domain IIIA), were studied in the absence and presence of Cuc D, E, and I, by fluorescence spectroscopy. The values of binding constant K(a) and binding site number n, determined by Scatchard method, increased for the both ligands only in the presence of Cuc E and D. Cuc I decreased slightly the K(a) of ibuprofen, suggesting an interaction with the domain IIIA of the protein. As a conclusion, Cuc E, D, and B produce rearrangement in the structure of albumin leading to increase the binding of domain specific ligands, ibuprofen and bilirubin.     

Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin

Streptococcal protein G (SpG) is a bacterial cell surface receptor exhibiting affinity to both human immunoglobulin (IgG) and human serum albumin (HSA). Interestingly, the serum albumin and immunoglobulin-binding activities have been shown to reside at functionally and structurally separated receptor domains. The binding domain of the HSA-binding part has been shown to be a 46-residue triple alpha-helical structure, but the binding site to HSA has not yet been determined. Here, we have investigated the precise binding region of this bacterial receptor by protein engineering applying an alanine-scanning procedure followed by binding studies by surface plasmon resonance (SPR). The secondary structure as well as the HSA binding of the resulting albumin-binding domain (ABD) variants were analyzed using circular dichroism (CD) and affinity blotting. The analysis shows that the HSA binding involves residues mainly in the second alpha-helix.     

Chiral recognition of metalaxyl enantiomers by human serum albumin: evidence from molecular modeling and photophysical approach

Metalaxyl is an acylamine fungicide, belonging to the most widely known member of the amide group. This task is aimed to scrutinize binding region and spatial structural change of principal vector human serum albumin (HSA) complex with (R)-/(S)-metalaxyl by exploiting molecular modeling, steady-state and time-resolved fluorescence, and circular dichroism (CD) approaches. According to molecular modeling, (R)-metalaxyl is situated within subdomains IIA and IIIA and the affinity of site I with (R)-metalaxyl is greater than site II, whereas (S)-metalaxyl is only located at subdomain IIA and the affinity of (S)-metalaxyl with site I is superior compared with that with (R)-metalaxyl. This coincides with the competitive ligand binding, guanidine hydrochloride-induced unfolding of protein, and hydrophobic 8-anilino-1-naphthalenesulfonic acid experiments; the acting forces between (R)-/(S)-metalaxyl and HSA are hydrophobic, π-π interactions, and hydrogen bonds, as derived from molecular modeling. Fluorescence emission manifested that the complex of (R)-/(S)-metalaxyl to HSA is the formation of adduct with an affinity of 10(4) M(-1), which corroborates the time-resolved fluorescence that the static type was operated. Furthermore, the changes of far-UV CD spectra evidence the polypeptide chain of HSA partially unfolded after conjugation with (R)-/(S)-metalaxyl. Through this work, we envisage that it can offer central clues on the biodistribution, absorption, and bioaccumulation of (R)-/(S)-metalaxyl.     

Opsonizing effect of serum and albumin gland extracts on the elimination of human erythrocytes from the circulation of Helix pomatia

The removal of human erythrocytes of the A₁ and B types from the circulation of the gastropod Helix pomatia follows an exponential curve. The elimination of the nonself particles is apparently dependent on serum opsonins as preincubation of A₁ and B erythrocytes in Helix serum increases the rate of their clearance. This conclusion is supported by our finding that secondary doses of nonsensitized A₁ erythrocytes injected 12–19 hr after a similar primary dose are cleared very slowly; however, the clearance rate returns to normal if erythrocytes comprising the second dose are preincubated with Helix serum. Furthermore, the elimination of second-dose A₁ erythrocytes is strongly enhanced after their pretreatment with agglutinating extracts of the albumin glands from H. pomatia and Cepaea (Helix) nemoralis. On the other hand, no opsonizing effect was obtained by pre-incubating A₁ erythrocytes in the agglutinating extract of the sponge Aaptos papillata.     

Determination on the binding of thiadiazole derivative to human serum albumin: a spectroscopy and computational approach

4-[3-acetyl-5-(acetylamino)-2,3-dihydro-1,3,4-thiadiazole-2-yl]phenyl benzoate from the family of thiadiazole derivative has been newly synthesized. It has good anticancer activity as well as antibacterial and less toxic in nature, its binding characteristics are therefore of huge interest for understanding pharmacokinetic mechanism of the drug. The binding of thiadiazole derivative to human serum albumin (HSA) has been investigated by studying its quenching mechanism, binding kinetics and the molecular distance, r between the donor (HSA) and acceptor (thiadiazole derivative) was estimated according to Forster’s theory of non-radiative energy transfer. The Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) changes of temperature-dependent K_b was calculated, which explains that the reaction is spontaneous and exothermic. The microenvironment of HSA have also been studied using synchronous fluorescence spectroscopy, and the feature of thiadiazole derivative-induced structural changes of HSA have been carried using Fourier transform infrared spectroscopy and the Molecular modelling simulations explore the hydrophobic and hydrogen bonding interactions.     

Multi-spectroscopic and molecular modeling studies of interaction between two different angiotensin I converting enzyme inhibitory peptides from gluten hydrolysate and human serum albumin

The present study was carried out to characterize Angiotensin-converting enzyme (ACE) inhibitory peptides which are released from the trypsin hydrolysate of wheat gluten protein. The binding of two inhibitory peptide (P₄ and P₆) to human serum albumin (HSA) under physiological conditions has been investigated by multi-spectroscopic in combination with molecular modeling techniques. Time-resolved and quenching fluorescence spectroscopies results revealed that the quenching of HSA fluorescence by P₄ and P₆ in the binary and ternary systems caused HSA-peptides complexes formation. The results indicated that both peptides quenched the fluorescence intensity of HSA through a static mechanism. The binding affinities and number of binding sites were obtained for the HSA-peptides complexes. The circular dichroism (CD) data revealed that the presence of both peptides increased the α-helix content of HSA and induced the remarkable folding of the polypeptide of the protein. Therefore, the CD data determined that the protein structure has been stabilized in the percent of ACE inhibitory peptides in binary and ternary systems. The binding distances between HSA and both peptides were estimated by the Forster theory, and it was revealed that nonradiative energy transfer from HSA to peptides occurred with a high probability. ITC experiments reveal that, in the absence and presence of P₆, the dominant forces are electrostatic in binary and ternary systems. Furthermore, molecular modeling studies confirmed the experimental results. Molecular modeling investigation suggested that P₄ bound to the site IA and IIA of HSA in binary and ternary systems, respectively. This study on the interaction of peptides with HSA should prove helpful for realizing the distribution and transportation of food compliments and drugs in vivo, elucidating the action mechanism and dynamics of food compliments and drugs at the molecular level. It should moreover be of great use for understanding the pharmacokinetic and pharmacodynamic mechanism of the food compliments and drugs.     

Complete coding sequence and molecular analysis of hepatitis A virus from a chimpanzee with fulminant hepatitis

Background Hepatitis A virus (HAV) infects both humans and non‐human primates, in experimentally infected chimpanzees is typically milder than in humans. In 1982, Abe and Shikata reported a first case of a chimpanzee with fulminant hepatitis caused by spontaneous HAV infection, and the underlying mechanisms of the disease remain unknown. Methods To characterize denoted CFH‐HAV, we conducted cloning and near full‐length sequence analysis. Results Phylogenetic analyses of VP1‐2A and complete sequence comparison between various genotypes and the sample sequence showed clustering in genotype IB. Based on BLAST analysis, the sequence was most closely related to the wild‐type (HM175/WT) isolate. Amino acid and nucleic acid similarities were 99.8% and 94.41%, respectively. Conclusions The chimpanzee may have been infected with human HAV genotype IB. The substitutions in VP2, VP4, 2B, 2C, and 3D, which may enhance virus proliferation, contributed to disease severity culminating in fulminant hepatic failure.     

Quenching of fluorescence by meclizine,a probe study for structural and conformational changes in human serum albumin

The goal of this study was to investigate the interactions between meclizine (MEC) and human serum albumin (HSA) under physiological conditions by different spectroscopies and molecular modeling technique. The drug, MEC quenched the intrinsic fluorescence of HSA and the analysis of the results revealed that static quenching mechanism. The binding of MEC quenches the HSA fluorescence; stoichiometry was 1:1 interaction. Thermodynamic quantities were calculated at different temperatures suggested that hydrophobic and van der Waals interaction with HSA–MEC. The molecular distance, r, between donor and acceptor was estimated according to Forster’s theory of non-radiation energy transfer. CD and FT-IR studies confirm changes of secondary structure of HSA. Molecular docking studies validate MEC molecule interact to HSA in sub domain IIA.