lnterferon

Molecular formula: C860Hi353N229O255S9 Molecular weight: 19269.1 CAS Registry No.: 76543-88-9 (aA), 99210-65-8 (a2B), 98059-61-1 (gamma)

SAMPLE

Matrix: solutions Sample preparation: Dilute PCR product solution 1:5 with water, inject an 80 uX aliquot.
HPLC VARIABLES

Column: 35 X 4.6 2.5 |xm TSK DEAE-NPR (Perkin-Elmer) Mobile phase: Gradient. A was 25 mM pH 9.0 Tris-HCl buffer. B was 25 mM pH 9.0 TrisHCl buffer containing 1 M NaCl. A:B from 70:30 to 45:55 over 30 s, to 35:65 over 2.5 min, to 0:100 over 30 s, maintain at 0:100 for 30 s, return to initial conditions over 30 s, re-equilibrate for 30 s. Flow rate: 1 Injection volume: 80 Detector: UV 260
CHROMATOGRAM

Retention time: 2.2

REFERENCE
Zeillinger, R.; Schneeberger, C; Speiser, P.; Kury, F. Rapid quantitative analysis of differential PCR products by high-performance liquid chromatography. BioTechniques, 1993, 15, 89-95

SAMPLE

Matrix: solutions Sample preparation: Inject an aliquot of a solution in glycerol:20 mM pH 7.2 sodium phosphate buffer 30:70.
HPLCVARIABLES

Column: 150 X 3 Separon SGX C-18 glass column Mobile phase: Gradient. A was 1 M pyridine adjusted to pH 5.0 with acetic acid. B was npropanol. A:B 80:20 for 20 min, to 30:70 over 1 h. Flow rate: 0.25 Detector: UV 280; bioassay
CHROMATOGRAM

Retention time: 46
REFERENCE
Aboagye-Mathiesen, G.; Toth, RD.; Juhl, C; Norskov-Lauritsen, N.; Petersen, P.M.; Ebbesen, P. Purification of human placental trophoblast interferon by two-dimensional high performance liquid chromatography. Prep.Biochem., 1991, 21, 35-51

SAMPLE

Matrix: solutions
HPLCVARIABLES

Column: 600 X 7.8 10 |xm Protein Pak J125 (Waters)

Mobile phase: Propylene glycol: buffer 25:75 (Buffer was 20 mM pH 7.0 sodium phosphate buffer containing 500 mM sodium sulfate and 0.04% Tween 20.) Flow rate: 0.5 Detector: UV 214 KEYWORDS interferon-omegal; GPC REFERENCE
Adolf, G.R.; Maurer-Fogy, L; Kalsner, L; Cantell, K. Purification and characterization of natural human interferon omega 1. Two alternative cleavage sites for the signal peptidase. J.Biol.Chem., 1990, 265, 9290-9295

SAMPLE

Matrix: solutions
HPLCVARIABLES Column: 250 X 4.6 5 |xm Bakerbond WP C18 Mobile phase: Gradient. A was 0.1% trifluoroacetic acid in water. B was 0.1% trifluoroacetic acid in MeCN. A:B 80:20 for 2 min, to 32:68 over 24 min, maintain at 32:68 for 10 min. Column temperature: 30 Flow rate: 1 Detector: UV 214; UV 280
CHROMATOGRAM

Retention time: 22
KEYWORDS interferon-omega 1 REFERENCE
Adolf, G.R.; Maurer-Fogy, L; Kalsner, L; Cantell, K. Purification and characterization of natural human interferon omega 1. Two alternative cleavage sites for the signal peptidase. J.Biol.Chem., 1990, 265, 9290-9295

SAMPLE

Matrix: solutions
HPLCVARIABLES Column: 300 X 4 Nucleosil 5C18 Mobile phase: Gradient. MeCN: 0.1% trifluoroacetic acid from 35:65 to 55:45 over 40 min. Flow rate: 0.9 Detector: UV 210 CHROMATOGRAM Retention time: 20 (Mf-I), 22 (Mf-2), 24 (Ms) REFERENCE
Nakagawa, S.; Honda, S.; Sugino, H.; Kusumoto, S.; Sasaoki, K.; Nishi, K.; Kakinuma, A. Characterization of three species of Escherichia coli-derived human leukocyte interferon A separated by reversephase high-performance liquid chromatography. J.Interferon.Res., 1987, 7, 285-299

SAMPLE

Matrix: solutions

HPLCVARIABLES

Guard column: Whatman Copell ODS Column: 250 X 10 Synchropak C18 RP-P Mobile phase: Gradient. A was 0.025% trifluoroacetic acid in water. B was 0.025% trifluoroacetic acid in MeCN. A:B from 70:30 to 40:60 over 30 min. Flow rate: 2 Detector: UV 220
CHROMATOGRAM

Retention time: 26 (IFNoA)

OTHER SUBSTANCES

Simultaneous: other forms of interferon

REFERENCE
Felix, A.M.; Heimer, E.P.; Tarnowski, S.J. Analysis of different forms of recombinant human interferons by high-performance liquid chromatography. Methods EnzymoL, 1986, 119, 242-248

SAMPLE

Matrix: solutions
HPLCVARIABLES

Guard column: Whatman Copell ODS Column: 250 X 10 Synchropak C18 RP-P Mobile phase: Gradient. A was 0.025% trifluoroacetic acid in water. B was 0.025% trifluoroacetic acid in MeCN. A:B from 70:30 to 40:60 over 30 min. Flow rate: 2 Detector: UV 220
CHROMATOGRAM

Retention time: 26 (IFNoA)

REFERENCE
Felix, A.M.; Heimer, E.P.; Lambros, T.J.; Swistok, J.; Tarnowski, S.J.; Wang, C-T. Analysis of different forms of recombinant human leukocyte interferons and synthetic fragments by high-performance liquid chromatography. J.Chromatogr., 1985, 327, 359-368

SAMPLE

Matrix: solutions Sample preparation: Centrifuge at 5000 g at 4 for 10 min, inject an aliquot.
HPLCVARIABLES

Column: 50 X 5 10 \xm Mono-S HPLC cation-exchange column (Pharmacia) Mobile phase: Gradient. A was 10 mM pH 7.0 sodium phosphate in ethylene glycol: water 20:80. B was 10 mM pH 7.0 sodium phosphate + 400 mM NaCl in ethylene glycol: water 20:80. A:B 100:0 for 30 min then to 0:100 over 60 min. Flow rate: 0.5 Detector: bioassay
CHROMATOGRAM

Retention time: 70
KEYWORDS

crude mixtures

REFERENCE
Friedlander, J.; Fischer, D.G.; Rubinstein, M. Isolation of two discrete human interferon-gamma (immune) subtypes by high-performance liquid chromatography Anal.Biochem., 1984, 137, 115-119

SAMPLE

Matrix: solutions Sample preparation: Pump 125 mL of a solution of interferon in ethylene glycol: 50 mM sodium phosphate buffer containing 1 M NaCl 50:50 onto the column (which was previously equilibrated with 1 M NaCl in ethylene glycol: water 50:50) then start the gradient.
HPLC VARIABLES

Column: 300 X 4.6 10 \xm Chromegabond octyl Mobile phase: Gradient. A was pyridine:formic acid:isopropanol:n-butanol:water 8:8: 20:3.3:60.7. B was pyridine: formic acid: isopropanol: n-butanol: water 8:8:25:20:39. A: B from 100:0 to 75:25 over 30 min, to 45:55 over 190 min, to 0:100 over 20 min, maintain at 0:100 for 1 h. Flow rate: 0.45 Injection volume: 125000 Detector: F; bioassay
CHROMATOGRAM

Retention time: 100

REFERENCE
Friesen, H.J.; Stein, S.; Pestka, S. Purification of human fibroblast interferon by high-performance liquid chromatography. Methods EnzymoL, 1981, 78, 430-435

SAMPLE

Matrix: solutions
HPLCVARIABLES

Column: MN-cyanopropyl Mobile phase: Gradient. Pyridine:formic acid:n-propanol:water from 8:8:0:84 to 8:8: 40:44 over 1 h. Flow rate: 0.3 Detector: F; bioassay
CHROMATOGRAM

Retention time: 40
REFERENCE
Friesen, H. J.; Stein, S.; Pestka, S. Purification of human fibroblast interferon by high-performance liquid chromatography. Methods EnzymoL, 1981, 78, 430-435

4
ANNOTATED BIBLIOGRAPHY

Feng, W.; Geng, X. Studies on silica-bonded monoclonal antibody packing material for separation of recombinant interferon by high performance immunoaffinity chromatography. Biomed.Chromatogr., 1993, 7, 317-320 [column was anti-interferon monoclonal antibody bonded to silica; gradient]