Virus elimination during the purification of monoclonal antibodies by column chromatography and additional steps

The theoretical potential for virus transmission by monoclonal antibody based therapeutic products has led to the inclusion of appropriate virus reduction steps. In this study, virus elimination by the chromatographic steps used during the purification process for two (IgG‐1 & ?3) monoclonal antibodies (MAbs) have been investigated. Both the Protein G (>7log) and ion‐exchange (5 log) chromatography steps were very effective for eliminating both enveloped and non‐enveloped viruses over the life‐time of the chromatographic gel. However, the contribution made by the final gel filtration step was more limited, i.e., 3 log. Because these chromatographic columns were recycled between uses, the effectiveness of the column sanitization procedures (guanidinium chloride for protein G or NaOH for ion‐exchange) were tested. By evaluating standard column runs immediately after each virus spiked run, it was possible to directly confirm that there was no cross contamination with virus between column runs (guanidinium chloride or NaOH). To further ensure the virus safety of the product, two specific virus elimination steps have also been included in the process. A solvent/detergent step based on 1% triton X‐100 rapidly inactivating a range of enveloped viruses by >6 log inactivation within 1 min of a 60 min treatment time. Virus removal by virus filtration step was also confirmed to be effective for those viruses of about 50 nm or greater. In conclusion, the combination of these multiple steps ensures a high margin of virus safety for this purification process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1341–1347, 2014     

Virus inactivation by protein denaturants used in affinity chromatography

Virus inactivation by a number of protein denaturants commonly used in gel affinity chromatography for protein elution and gel recycling has been investigated. The enveloped viruses Sindbis, herpes simplex-1 and vaccinia, and the non-enveloped virus polio-1 were effectively inactivated by 0.5 M sodium hydroxide, 6 M guanidinium thiocyanate, 8 M urea and 70% ethanol. However, pH 2.6, 3 M sodium thiocyanate, 6 M guanidinium chloride and 20% ethanol, while effectively inactivating the enveloped viruses, did not inactivate polio-1. These studies demonstrate that protein denaturants are generally effective for virus inactivation but with the limitation that only some may inactivate non-enveloped viruses. The use of protein denaturants, together with virus reduction steps in the manufacturing process should ensure that viral cross contamination between manufacturing batches of therapeutic biological products is prevented and the safety of the product ensured.     

Bovine renal glomerular basement membrane. Isolation and characterization of a glycoprotein component.

Bovine glomerular basement membrane was extracted with 6 M guanidinium chloride and the soluble material fractionated on a Bio-Gel A-1.5m column in 1% Na dodecyl-SO4. A single component was obtained by reduction of a selected column fraction with 2-mercaptoethanol followed by chromatography on an analytical Bio-Gel A-1.5m column and shown to be homogenous by electrophoresis and ultracentrifugation. It consists of 90% protein and 8.6% carbohydrate by weight. The amino acid composition is characterized by the presence of low amounts of hydroxyproline and hydroxylysine, and substantial amounts of aspartic acid, glutamic acid, half-cystine, and glycine. It contains all the monosaccharide constituents present in the whole basement membrane indicating the presence of both heteropolysaccharide and disaccharide units; the presence of the latter unit was demonstrated unequivocally by ion exchange chromatography. The component contains 1 heteropolysaccharide unit and 4 dissaccharide units/molecule of Mr equals 70,000. The molecular weight of component VII was determined by several methods. Molecular weight values of 68,000 +/- 3,000 and 72,000 +/- 2,000 were determined in 6 M guanidinium chloride by the methods of sedimentation equilibrium and gel filtration chromatography, respectively, and values of 136,000 +/- 3,100 and 140,000 +/- 2,000 were determined in 1% Na dodecyl-SO4 by the methods of polyacrylamide gel electrophoresis and gel filtration chromatography, respectively. Circular dichroism spectra indicate that component VII assumes a random coil conformation in 6 M guanidinium chloride and a more disordered conformation in 1% Na dodecyl-SO4 than standard proteins used in calibration of polyacrylamide gels and gel filtration column. These results indicate that the minimal molecular weight of component VII is about 70,000 and that the anomalous behavior in Na dodecyl-SO4 is due in part to its conformation.     

The interaction of sodium dodecyl sulfate with cucumber green mottle mosaic virus protein and tobacco mosaic virus protein

The binding of sodium dodecyl sulfate to coat protein subunits of cucumber green mottle mosaic virus and tobacco mosaic virus was studied by equilibrium dialysis. The amount of dodecyl sulfate bound to the cucumber virus protein in 0.1 m phosphate buffer (pH 7.2) was found to be 1.55 g/g, which was the same value as that obtained with the tobacco virus protein. The presence of 8 m urea markedly decreased the degree of binding of dodecyl sulfate to the proteins. The amount of binding to the cucumber virus protein was reduced to 0.56 g/g, and that to the tobacco virus protein decreased to 0.8 g/g. The net charges of both proteins were negative at neutral pH and the amount of negative charge of the cucumber virus protein, obtained from the potentiometric titration curves, was larger than that of the tobacco virus protein, either in the native state or in the denatured state. In dodecyl sulfate/polyacrylamide gel electrophoresis the cucumber virus protein migrated faster than the tobacco virus protein. On the other hand, in the presence of 8 m urea, the electrophoretic migration rate of the cucumber virus protein was equal to that of the tobacco virus protein. Sedimentation equilibrium experiments in 6 m guanidinium chloride gave molecular weights of 17,700 and 17,200 for the tobacco mosaic virus and the cucumber virus proteins, respectively. These results suggest that the effective negative charge density of the cucumber virus protein-dodecyl sulfate complex is higher than that of the tobacco virus proteindodecyl sulfate complex in 0.1% dodecyl sulfate solution. The conformation of both proteins was investigated by circular dichroism measurements. Both proteins have a slightly higher degree of α-helix content in dodecyl sulfate solution than in the native state. The addition of 8 m urea to both proteins while in this solution induced a change in conformation to one having a much smaller degree of ordered structure, although the change in the cucumber virus protein was more intense than that in the tobacco virus protein.     

The use of gel filtration to follow conformational changes in proteins. Conformational flexibility of bovine myelin basic protein.

The hydrodynamic behavior of bovine myelin basic protein was studied by gel filtration through Sephadex G-100 under conditions which included variations in pH from 2 to 12, variations in ionic strength from 0.01 to 1.5 M at pH 2 and from 0.1 to 2 M at pH 7, and variations in guanidinium chloride concentration from 0 to 6 M. A number of well characterized compact globular proteins were subjected to the same conditions for comparison. Compact globular proteins showed major conformational transitions due to acid, alkali, and guanidinium chloride denaturation and, possibly, minor transitions as well. Myelin basic protein behaved like a flexible linear polyelectrolyte, expanding continuously between pH 11 and pH 2 to 3 at ionic strength 0.1 M and contracting continuously with increase in ionic strength at pH 2 and at pH 7 to the point of salting-out. Relatively low concentrations of guanidinium chloride (approximately 0.5 M) were sufficient to cause the basic protein to expand. With increasing concentration of the denaturant the molecule continued to expand, but in a noncooperative manner. These results demonstrated the lack of significant intramolecular stabilization in the protein.     

Simultaneous isolation of major viral proteins in one step.

We have developed a rapid and simple technique for the simultaneous isolation of all the major viral proteins from RNA tumor viruses. The basis for this procedure is analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using dansylated virus as internal marker it is possible to follow the migration of unlabeled viral proteins since dansylation does not change the mobility of labeled proteins (8). The method results in approximately 80% recovery of starting protein and is very reproducible. Using radioimmunoassay no alteration of the purified proteins is detectable.     

Dilatometric studies of the denaturation of immunoglobulin G by guanidinium chloride

The denaturation of immunoglobulin G and its light chains by guanidinium chloride at 25°C was followed using the dilatometric method. From results of the dilatometric measurements the differences between the partial molar volumes of the proteins in water and in guanidinium chloride solutions of various concentrations, respectively, have been obtained. The differences reflect the extent of unfolding as well as the denaturant binding to the protein. Several experiments were also performed in which the protein disulfide bonds were reduced by dithioerythritol.The volume change produced by the reduction of one mole of disulfide bonds of immunoglobulin G in 6 M guanidinium chloride was found to be the same as that for oxidized glutathione; the value was ?0.9 ml/mol.     

Type-C RNA Virus Gene Expression in Human Tissue

Partially purified fractions of human tissues have been analyzed by competition radioimmunoassay for the presence of two of the principle structural components of type-C RNA viruses, the major core protein (p27 to p30) and the major envelope glycopeptides (gp69/71). Screening of tissues was carried out by use of a heterologous assay system of (125)I-labeled Rauscher murine virus p30 antigen and anti-RD 114 virus serum which was found to detect a class of interspecies determinants common to murine, feline, and primate viruses. A competitor with the same apparent affinity for antibody binding as that of purified viral core proteins was found in relatively high concentration in tissues from patients with systemic lupus erythematosus, in some neoplastic tissues, and also in normal human tissues. This competitor from a lupus spleen chromatographed on phosphocellulose and showed size fractionation during gel filtration similar to known p27 to p30 viral proteins. An immunologically reactive protein was also demonstrated by immunodiffusion and by immunoprecipitation of (125)I-labeled human protein with anti-RD 114 p28 serum. Analysis of these human competitor proteins with homologous assay systems of viral core proteins and corresponding antisera showed that all, including the normal tissue extracts, appear similar to core proteins of known viruses, especially the RD 114 and woolly monkey species. A hypothesis suggested by these data is that many, if not all, humans harbor at least part of the genome of one or more type-C viruses, the properties of which are similar to those of viruses from other mammalian species, particularly primates.     

Virions and intracellular nucleocapsids produced during mixed heterotypic influenza infection of MDCK cells.

Phenotypically mixed virus yields, obtained by coinfection of MDCK cells with influenza A/WSN/33 and B/Lee/40 viruses, contained both A/WSN/33 and B/Lee/40 NP proteins, as revealed by polyacrylamide gel electrophoresis of the purified 14C-amino acids-labeled virus. Virions were lysed with 0.6 M KCl-Triton X-100 buffer, and nucleocapsids were immunoprecipitated with antibodies against NP protein of influenza A virus. Polyacrylamide gel electrophoresis of the immunoprecipitate revealed NP protein of A/WSN/33 but not of B/Lee/40 virus. However, in similar experiments with the lysates of doubly infected cells, the band of B/Lee/40 NP protein was revealed in the polyacrylamide gel electrophoresis patterns of the immunoprecipitates. In an attempt to analyze the RNA content of the immune complexes, we absorbed the lysates of doubly infected [3H]uridine-labeled cells with protein A-containing Staphylococcus aureus covered with antibodies against the NP protein of influenza A virus. RNA extracted from the immune complexes contained genomic RNA segments of both A/WSN/33 and B/Lee/40 viruses. In control samples containing an artificial mixture of cell lysates separately infected with each virus, the analysis revealed homologous components (i.e., A/WSN/33 NP protein or RNA segments) in the immune complexes. The results suggest the presence of phenotypically mixed nucleocapsids in the cells doubly infected with influenza A and B viruses; in the course of the virion formation, the nucleocapsids lacking the heterologous NP protein are selected.     

Evolution and Taxonomy of Positive-Strand RNA Viruses: Implications of Comparative Analysis of Amino Acid Sequences

Abstract

Despite the rapid mutational change that is typical of positive-strand RNA viruses, enzymes mediating the replication and expression of virus genomes contain arrays of conserved sequence motifs. Proteins with such motifs include RNA-dependent RNA polymerase, putative RNA helicase, chymotrypsin-like and papain-like proteases, and methyltransferases. The genes for these proteins form partially conserved modules in large subsets of viruses. A concept of the virus genome as a relatively evolutionarily stable “core” of housekeeping genes accompanied by a much more flexible “shell” consisting mostly of genes coding for virion components and various accessory proteins is discussed. Shuffling of the “shell” genes including genome reorganization and recombination between remote groups of viruses is considered to be one of the major factors of virus evolution.

Multiple alignments for the conserved viral proteins were constructed and used to generate the respective phylogenetic trees. Based primarily on the tentative phylogeny for the RNA-dependent RNA polymerase, which is the only universally conserved protein of positive-strand RNA viruses, three large classes of viruses, each consisting of distinct smaller divisions, were delineated. A strong correlation was observed between this grouping and the tentative phylogenies for the other conserved proteins as well as the arrangement of genes encoding these proteins in the virus genome. A comparable correlation with the polymerase phylogeny was not found for genes encoding virion components or for genome expression strategies. It is surmised that several types of arrangement of the “shell” genes as well as basic mechanisms of expression could have evolved independently in different evolutionary lineages.

The grouping revealed by phylogenetic analysis may provide the basis for revision of virus classification, and phylogenetic taxonomy of positive-strand RNA viruses is outlined. Some of the phylogenetically derived divisions of positive-strand RNA viruses also include double-stranded RNA viruses, indicating that in certain cases the type of genome nucleic acid may not be a reliable taxonomic criterion for viruses.

Hypothetical evolutionary scenarios for positive-strand RNA viruses are proposed. It is hypothesized that all positive-strand RNA viruses and some related double-stranded RNA viruses could have evolved from a common ancestor virus that contained genes for RNA-dependent RNA polymerase, a chymotrypsin-related protease that also functioned as the capsid protein, and possibly an RNA helicase.     

Extremely Stable Soluble High Molecular Mass Multi-Protein Complex with DNase Activity in Human Placental Tissue

Human placenta is an organ which protects, feeds, and regulates the grooving of the embryo. Therefore, identification and characterization of placental components including proteins and their multi-protein complexes is an important step to understanding the placenta function. We have obtained and analyzed for the first time an extremely stable multi-protein complex (SPC, ∼1000 kDa) from the soluble fraction of three human placentas. By gel filtration on Sepharose-4B, the SPC was well separated from other proteins of the placenta extract. Light scattering measurements and gel filtration showed that the SPC is stable in the presence of NaCl, MgCl2, acetonitrile, guanidinium chloride, and Triton in high concentrations, but dissociates efficiently in the presence of 8 M urea, 50 mM EDTA, and 0.5 M NaCl. Such a stable complex is unlikely to be a casual associate of different proteins. According to SDS-PAGE and MALDI mass spectrometry data, this complex contains many major glycosylated proteins with low and moderate molecular masses (MMs) 4–14 kDa and several moderately abundant (79.3, 68.5, 52.8, and 27.2 kDa) as well as minor proteins with higher MMs. The SPC treatment with dithiothreitol led to a disappearance of some protein bands and revealed proteins with lower MMs. The SPCs from three placentas efficiently hydrolyzed plasmid supercoiled DNA with comparable rates and possess at least two DNA-binding sites with different affinities for a 12-mer oligonucleotide. Progress in study of placental protein complexes can promote understanding of their biological functions.     

Complex structure of human bronchial mucus glycoprotein

Human bronchial mucus glycoproteins or mucins were isolated from the sputum of two patients by a method avoiding reducing agents and involving water extraction and gel filtration on Sepharose CL-2B in 6 M guanidinium chloride. The chemical analysis indicated approximately 25-40% lipid. The amino acid and carbohydrate analysis differ quantitatively from that of mucins purified after prior reduction of mucus. These fractions also have a higher proportion of aspartic and glutamic acids than that of the mucins from reduced sputum. These mucins are still contaminated by small amounts of peptides but do not seem to contain disulfide-attached cross-linking protein. Human bronchial mucins have a strong tendency to form aggregates except in 6 M guanidinium chloride. Electron microscopy performed with various procedures indicates the presence of both micelles and flexible threads measuring 200-1000 nm. Delipidation removes most of the micellar forms. Thereafter mucins appear mainly as polydisperse flexible extended threads and also as aggregates. These features of bronchial mucins do not fit with the generally accepted idea of mucin subunits linked by disulfide bridges (unless they are linked end to end) and alternatively favour a model where mucin molecules behave like filaments that could easily aggregate according to the solvent system (mucin concentration, absence of dissociating conditions).     

Specific binding of the type C viral core protein p12 with purified viral RNA.

The major viral phosphoproteins (p12) of the Rauscher murine leukemia virus (R-MuLV) and the simian sarcoma-associated virus (SSAV) bind in vitro to their homologous 70S and 35S viral RNAs. Using purified 32P-labeled RNA and 125I-labeled p12 protein, complexes that are stabilized by formaldehyde-cross-linking can be readily detected after velocity gradient centrifugation. The in vitro reconstructed ribonucleoprotein complexes are seen only with p12 proteins incubated with viral RNAs isolated from the same type C viruses; no such complexes form with heterologous protein-RNA mixtures. Homologous but not heterologous p12 molecules compete with radiolabeled p12 protein for the specific viral RNA binding sites. The competition assay permits the detection of 10 ng of viral p12 protein. The major internal protein of type C viruses (p30) does not bind to viral RNA using identical assay conditions. From the specific activities of the radiolabeled components and also by equilibrium sedimentation analysis, we estimate that fewer than 15 molecules of p12 protein bind to each molecule of viral RNA. Both the specificity and stoichiometry of the p12-RNA interactions suggest that these RNA tumor virus proteins have a regulatory role in cells.     

Chromatographic separation and antigenic analysis of proteins of the oncornaviruses. V. Identification of a new murine viral protein, p15(E).

Profiling of murine leukemia virus (MuLV) proteins by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) has revealed a low-molecular-weight protein which does not appear in the corresponding region of viral protein profiles obtained by gel filtration in 6 M guanidine hydrochloride. This protein species, termed p15(E), is easily demonstrable in MuLV isolates for which the viral p15 and p12 proteins have almost identical electrophoretic mobilities; this leaves a protein slightly larger than these two in the PAGE system unaccounted for in the gel filtration system. However, antiserum against the void volume fraction of the gel filtration eluate precipitated the p15(E) component from solubilized, radiolabeled virions, as shown by SDS-PAGE analysis of such immunoprecipitates. Comparative radioprecipitation analyses of this type revealed that for various MuLV isolates p15(E) was distinguishable from p15 in terms of serological reactivities, relative mobilities in gel electrophoresis, and relative efficiencies of labeling with individual amino acids. Thus it appears that, as is the case for avian oncornaviruses, MuLVs contain seven major structural proteins.     

High yield refolding and purification process for recombinant human interleukin-6 expressed in Escherichia coli

Recombinant human interleukin-6 (hIL-6), a pleiotropic cytokine containing two intramolecular disulfide bonds, was expressed in Escherichia coli as an insoluble inclusion body, before being refolded and purified in high yield providing sufficient qualities for clinical use. Quantitative reconstitution of the native disulfide bonds of hIL-6 from the fully denatured E. coli extracts could be performed by glutathione-assisted oxidation in a completely denaturating condition (6M guanidinium chloride) at protein concentrations higher than 1 mg/mL, preventing aggregation of reduced hIL-6. Oxidation in 6M guanidinium chloride (GdnHCl) required remarkably low concentrations of glutathione (reduced form, 0.01 mM; oxidized form, 0.002 mM) to be added to the solubilized hIL-6 before the incubation at pH 8.5, and 22 degrees C for 16 h. After completion of refolding by rapid transfer of oxidized hIL-6 into acetate buffer by gel filtration chromatography, residual contaminants including endotoxin and E. coli proteins were efficiently removed by successive steps of chromatography. The amount of dimeric hIL-6s, thought to be purification artifacts, was decreased by optimizing the salt concentrations of the loading materials in the ion-exchange chromatography, and gradually removing organic solvents from the collected fractions of the preparative reverse-phase HPLC. These refolding and purification processes, which give an overall yield as high as 17%, seem to be appropriate for the commercial scale production of hIL-6 for therapeutic use.     

Concerning the use of empirical methods for determining the molecular weight of a small glycopeptide cleaved from bovine rhodopsin

The molecular weight of a glycopeptide cleaved enzymatically from bovine rhodopsin was investigated by a variety of techniques, such as gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of urea, and gel filtration in 6 guanidinium chloride after reduction and carboxymethylation. Great variation in the estimation of the molecular weight between the several techniques was observed. When compared to an estimate of 9000 based on amino acid analysis and considerations of its carbohydrate content, values ranging between 4500 and 18,000 were obtained by the other methods. Thus, while the molecular weight of the glycopeptide remains unresolved, the resultant ambiguity in evaluating this property may exemplify the limitations inherent in applying empirical methods to the determination of the molecular weight of small glycopeptides.     

Structural components of influenza C virions.

The genome RNA species of influenza type C virions were analyzed by polyacrylamide gel electrophoresis. The pattern obtained was found to resemble those of other influenza viruses. Six RNA species were resolved, with estimated sizes ranging from 0.37 X 10(6) to 1.25 X 10(6) daltons. The internal ribonucleoproteins of influenza C virions were found to sediment heterogeneously in glycerol velocity gradients as demonstrated previously with influenza A/WSN virus. The ribonucleoproteins possessed diameters of 12 to 15 nm, with lengths ranging from 30 to 100 nm. Of the three major virion polypeptides (molecular weights, 88,000, 66,000, and 26,000), only the largest is glycosylated. Similar polypeptide species were present in influenza C virions of five different strains. All three major proteins of influenza C virions possess electrophoretic mobilities distinguishable from those of the major polypeptides of influenza A/WSN. The 66,000-dalton protein is associated with the ribonucleoprotein components. Two additional glycosylated polypeptides, with estimated molecular weights of 65,000 and 30,000, were detected in virions grown in embryonated eggs, but not in virus particles obtained from chicken embryo fibroblasts.