Purification Of Human Fibroblast Interferon Research Articles

4658017 Method for the large scale purification of human fibroblast interferon: Wlodzimierz E Dembinski, Eugene Sulkowski assigned to Health Research Inc (Roswell Park Division)

4658017 Method for the large scale purification of human fibroblast interferon: Wlodzimierz E Dembinski, Eugene Sulkowski assigned to Health Research Inc (Roswell Park Division)

Human interferon for clinical trials: removal of pyrogen by a simple two-step procedure.

Commonly used preparations of human interferon are pyrogenic when used in humans for clinical trials. We describe a simple two-step procedure for the purification of human fibroblast interferon, employing Blue Sepharose chromatography and high-speed centrifugation. Such preparations are devoid of pyrogenic activity in a rabbit test system and are much more suitable for use in human clinical trials than previously used preparations.

Purification of Human Fibroblast Interferon by Zinc Chelate Chromatography

Human interferon was prepared by superinduction of cultures of either diploid embryonic skin and muscle cells or of the osteosarcoma cell line MG-63. The interferon so obtained was concentrated and partially purified by adsorption to controlled pore glass (CPG) beads at neutral pH and desorption by glycine-HCl buffer at pH 2. After neutralization, this interferon was applied to a column of zinc chelate which was eluted with buffers of decreasing pH. Most of the proteins eluted ahead of the interferon activity, which itself eluted in two distinct peaks. The first peak occurred in the effluent fractions around pH 5.9, and the second one in fractions around pH 5.2. The interferon found in fractions of pH 5.9 contained 5% of the original contaminating proteins. In contrast, the amount of total protein in the pH 5.2 peak was so small that it could not accurately be assayed by the fluorescamine method. Consequently, the interferon in the peak fraction was estimated to have a specific activity of about 2 x 10(9) units/mg. This material was radiolabelled and analysed by electrophoresis. A major peak of about 22000 mol. wt. with only minor contaminating proteins appeared on the autoradiographs. The total recovery of the zinc chelate chromatographical procedure was nearly 100%, and the interferon recovered from each peak behaved consistently on rechromatography. Fibroblast interferon produced by most diploid cells contained less than 10% of the variant eluting at pH 5.9. MG-63 cells and high-passage cultures of some diploid cell strains produced up to 50% of this variant.

63] Purification of human fibroblast interferon by high-performance liquid chromatography

This chapter presents a procedure combining the techniques of affinity chromatography and high-performance liquid chromatography (HPLC) for the preparation of fibroblast interferon from serum-containing medium. The purified fibroblast interferon is homogeneous by several criteria of molecular characterization and has a specific activity of approximately 4 x l0 8 units/mg. The purified protein is present in a completely volatile, detergent-free solvent. From several possible approaches, the combination of Blue Dextran-Sepharose affinity chromatography with reverse-phase HPLC at acidic pH is outlined. In contrast to leukocyte interferon, it is not possible to use neutral pH conditions for the HPLC steps because fibroblast interferon rapidly lost activity when organic solvents (propanols, acetone, Methylcellosolve, and such) are present. The data for the homogeneous protein is a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, which coincided with the recoverable antiviral activity, and a single protein peak in the last chromatographic step, which coincided with the antiviral activity. The purified preparations are characterized by amino acid analyses, amino sugar analyses, end-group determinations, tryptic maps, and amino acid sequencing.

61] Purification of human fibroblast interferon prepared in the absence of serum

Human fibroblast interferon is purified to homogeneity, and all started with interferon produced in the absence of serum. This contribution describes a recent procedure for the purification to homogeneity of large volumes of human fibroblast interferon produced in the absence of added serum. Interferon is assayed by a microtiter technique by the inhibition of vesicular stomatitis virus cytopathic effect as a measure of the interferon concentration. When this interferon is subjected to a second electrophoresis and the proteins are stained with Coomassie Blue, the data are obtained. Only one band is observed, indicating homogeneous interferon. A more vigorous proof of purity is to determine the number of peptide chains in a fraction of purified interferon. This can be done on microgram quantities of a protein by Edman degradation starting at the NH 2 terminus. More than one NH 2 -terminal amino acid sequence indicates more than one protein. The NH 2 -terminal sequence of fibroblast interferon purified by the procedure has been determined by Edman degradation. Only one sequence is found, proving that this purification procedure gives pure human fibroblast interferon.

64] Purification of human fibroblast interferon produced in the absence of serum by cibacron blue F3GA-agarose and high-performance liquid chromatography

This chapter describes the procedure that combines the use of affinity chromatography and high-performance liquid chromatography to purify human fibroblast interferon produced in the absence of serum. The final product has a specific activity of about 3 x 10 8 units per milligram of protein and is homogeneous by the criteria of sodium dodecyl sulfate (SDS)-gel electrophoresis, amino acid analysis, and amino acid sequencing. In addition, this procedure has the advantage that the pure interferon is left in a volatile buffer, and thus is easily concentrated or dried for further studies. Affinity chromatography provides a high purification factor, but yields a dilute solution of interferon in 50% ethylene glycol. The final product is then obtained in concentrated form by HPLC. The interferon may be recovered salt- and solvent- free by evaporation because only volatile eluents are used. As proof of homogeneity, a single band is obtained on SDS-polyacrylamide gel electrophoresis as well as a single NH2-terminal amino acid sequence.

NH2-terminal amino acid sequence of human fibroblast interferon.

The purification of human fibroblast interferon by chromatography on Blue Sepharose and high-performance liquid chromatography is described. The amino acid composition and a partial sequence of the homogeneous protein are reported. The NH2 terminus was determined to be NH2-Met1-Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser-Asn-Phe-Gln-X-Gln-Lys.

Human fibroblast interferon: amino acid analysis and amino terminal amino acid sequence.

The purification of human fibroblast interferon has been simplified to a two-step procedure consisting of affinity chromatography on Blue Sepharose and sodium dodecyl sulfate polyacrlamide gel electrophoresis. A preliminary amino acid composition and the sequence of the 13 amino-terminal residues of homogeneous interferon prepared by this method is reported.

Human fibroblast interferon for clinical trials: production, partial purification, and characterization.

The production and partial purification of human fibroblast interferon for performing clinical trials is described. The interferon was produced by superinduction (exposure to riboinosinic-ribocytidylic acid, cycloheximide, and actinomycin D) of large numbers of human diploid fibroblast cultures. The yield averaged 750 units per cm(2) of culture area. The interferon was concentrated and purified by a two-step procedure involving acid desorption from controlled-pore glass beads and dialysis against polyethylene glycol. Human plasma protein was added as a stabilizer. The lyophilized end product had a specific activity of 0.5 x 10(6) to 1 x 10(6) units/mg of protein; it could be reconstituted for injection at a concentration of 2 x 10(6) units/ml. The composition of this interferon was characterized by crossed immunoelectrophoresis with polyspecific antibodies prepared against the principal sources of potential contaminants: human serum, calf serum, and normal human fibroblasts. Several components of each source were detected. Although the major component of calf serum, bovine serum albumin, was absent, other minor components were retained by the production and purification sequence. One of the main contaminants of fibroblast origin was found to be fibronectin.

Purification of human diploid fibroblast interferon by immobilized neuraminidase.

An efficient method for the purification of human fibroblast interferon (IF) based on binding via the N-acetyl neuraminic acid (N-ANA) residue of the IF molecules to the immobilized neuraminidase has been developed. Binding of IF occurred at pH 4.5 and elution of the bound activity was effected at pH 9.5. Specific activity of IF in the alkaline eluate was increased by a factor in excess of 200 and the IF thus recovered had probably retained most of the N-ANA moieties.

Purification of human fibroblast interferon by zinc chelate affinity chromatography.

Zinc chelated by iminodiacetic acid linked to an insoluble matrix binds human fibroblast interferon quite selectively at neutral pH in 0.15 M NaCL. On reduction of the pH and increase of the ionic strength, the interferon is eluted. Using a pH gradient at constant high ionic strength, good purification of the interferon can be obtained, up to a final specific activity of 108.5 units/mg of protein. Approximately 60% of the applied activity can be recovered.

Binding of human fibroblast interferon to concanavalin A-agarose. Involvement of carbohydrate recognition and hydrophobic interaction.

Human fibroblast interferon binds to a concanavalin A-agarose (Con A-Sepharose) equilibrated with methyl alpha-D-mannopyranoside, or levan; in contrast, it is only partially retarded on a similar column equilibrated with ethylene glycol. Interferon does not bind, however, to a lectin column equilibrated with both methyl alpha-D-mannopyranoside and ethylene glycol. Thus, a hydrophobic interaction between fibroblast interferon and the immobilized lectin seems to account for a large portion of the binding forces involved. Other hydrophobic solutes, such as dioxane, 1, 2-propanediol, and tetraethylammonium chloride, were found equally or more efficient than ethylene glycol in displacing interferon from the lectin column. The elution pattern of interferon from a concanavalin A-agarose (Con A-Sepharose) column, at a constant ehtylene glycol concentration and with an increasing mannoside concentration, reveals the existence of four distinct interferon components. The selective adsorption to and elution from a concanavalin A-agarose (Con A-Sepharose) column resulted in about a 3000-fold purification of human fibroblast interferon and complete recovery of activity. The specific activity of the partially purified interferon preparation is about 5 X 10(7) units per mg of protein. The chromatographic behavior of human leukocyte interferon is remarkable in that it does not bind to concanavalin A-agarose at all indicating the absence of carbohydrate moieties recognizable by the lectin, or if present, their masked status. When concanavalin A was coupled to an agarose matrix (cyanogen bromide activated) at pH 8.0 and 6.0 human fibroblast interferon bound to both lectin-agarose adsorbents and could be recovered with methyl alpha-D-mannopyranoside. Concanavalin A, immobilized directly on agarose matrix at pH 8.0 and 6.0, thus displays only carbohydrate recognition toward interferon. By contrast, unless a hydrophobic solute was included in the solvent containing methyl mannoside, human fibroblast interferon could not be recovered from concanavalin A-agarose coupled at pH 9.0. When concanavalin A was immobilized via molecular arms, in tetrameric as well as dimeric forms, the binding of interferon again occurred exclusively through carbohydrate recognition. Thus, the hydrophobic interaction can be eliminated by appropriate immobilization of the lectin, and then adsorbed glycoproteins, as exemplified here by interferon, can be recovered readily with methyl mannoside alone.