Somatropin

Molecular formula: C 990 H 1528 N 262 O 300 S 7

Molecular weight: 21500.0
CAS Registry No.: 9002-72-6, 12629-01-5 (human)

SAMPLE
Matrix: formulations
Sample preparation: Prepare a 1 mg/mL solution in 12.5 mM ammonium bicarbonate,
filter (0.45 |xm), inject an aliquot.

HPLCVARIABLES
Guard column: 40 X 6 TSK gel SWXL (Tosoh)
Column: 300 X 7.8 TSK gel G2000SWXL
Mobile phase: 50 mM pH 6.8 Phosphate buffer containing 300 mM NaCl and 0.05% sodium
azide (Caution! Sodium azide is carcinogenic, highly toxic, and can form explosive com-
pounds if discharged to the sewer!)
Flow rate: 0.8
Injection volume: 20
Detector: UV 280; RI

CHROMATOGRAM
Retention time: 12.5

OTHER SUBSTANCES
Simultaneous: impurities

KEYWORDS
recombinant; GPC
REFERENCE
Kawashige, M.; Sendo, T.; Otsubo, K.; Aoyama, T.; Oishi, R. Quality evaluation of commercial lyophilized
human growth hormone preparations. Biol.Pharm.BulL, 1995, 18, 1793-1796

SAMPLE
Matrix: solutions

HPLCVARIABLES
Column: Zorbax GF-250
Mobile phase: 50 mM ammonium bicarbonate
Flow rate: 0.6
Detector: UV 215

CHROMATOGRAM
Retention time: 13.5, 14.5

KEYWORDS
SEC
REFERENCE
Katakam, M.; Bell, L.N.; Banga, A.K. Effect of surfactants on the physical stability of recombinant
human growth hormone. J.Pharm.Sci., 1995, 84, 713-716
SAMPLE
Matrix: solutions

HPLCVARIABLES
Guard column: 30 X 4.6 10 |xm RP 300 Aquapore MPLC (Brownlee)
Column: 100 X 4.6 10 |xm RP 300 Aquapore MPLC (Brownlee)
Mobile phase: Gradient. A was 0.1% trifluoroacetic acid in water. B was 0.1% trifluoroacetic
acid in MeCN. A:B from 54:46 to 43:57 over 22 min.
Detector: UV 214

KEYWORDS
recombinant; cow
REFERENCE
Stevenson, CL.; Hageman, M.J. Estimation of recombinant bovine somatropin solubility by excluded-
volume interaction with polyethylene glycols. Pharm.Res., 1995, 12, 1671 1676

SAMPLE
Matrix: solutions

HPLC VARIABLES
Column: 250 X 4.6 5 |jim Vydac C4
Mobile phase: Gradient. MeCN: 0.05% trifluoroacetic acid from 43:57 to 55:45 over 30 min.
Column temperature: 45
Flow rate: 0.5
Injection volume: 20
Detector: UV 220

CHROMATOGRAM
Retention time: 28

OTHER SUBSTANCES
Simultaneous: somatrem

REFERENCE
Arcelloni, C; Fermo, L; Banfi, G.; Pontiroli, A.E.; Paroni, R. Capillary electrophoresis for protein
analysis: separation of human growth hormone and human insulin molecular forms. Anal.Biochem.,
1993,222, 160-167

SAMPLE
Matrix: solutions

HPLCVARIABLES
Column: 600 X 7.5 10 |xm TSK G3000 SW (Pharmacia)
Mobile phase: 10 mM pH 7.2 K2HPO4 containing 300 mM NaCl
Flow rate: 1
Injection volume: 20
Detector: UV 254

CHROMATOGRAM
Retention time: 17 (44K), 21 (22K)
REFERENCE
Arcelloni, C; Fermo, L; Banfi, G.; Pontiroli, A.E.; Paroni, R. Capillary electrophoresis for protein
analysis: separation of human growth hormone and human insulin molecular forms. Anal.Biochem.,
1993,222, 160-167
SAMPLE
Matrix: solutions

HPLCVARIABLES
Column: 250 X 4.6 5 jxm C4 (Vydac)
Mobile phase: Isopropanol: 500 mM pH 6.5 KH2PO4 29:71
Column temperature: 45
Flow rate: 1
Detector: F ex 295 em 348

CHROMATOGRAM
Retention time: 25

OTHER SUBSTANCES
Simultaneous: somatrem

REFERENCE
Oroszlan, P.; Wicar, S.; Teshima, G.; Wu, S.L.; Hancock, W.S.; Karger, B.L. Conformational effects in the
reversed-phase chromatographic behavior of recombinant human growth hormone (rhGH) and N-
methionyl recombinant human growth hormone (Met-hGH). Anal.Chem., 1992, 64, 1623-1631

SAMPLE
Matrix: solutions
Sample preparation: Dissolve in 100 mM NaH2PO4 adjusted to pH 2.1 with orthophos-
phoric acid, inject a 100 u,L aliquot.

HPLC VARIABLES
Column: 250 X 4 Aquapore RP 300 (Kontron)
Mobile phase: Gradient. A was 100 mM NaH2PO4 adjusted to pH 2.1 with orthophosphoric
acid. B was MeOH. A:B from 90:10 to 35:65 over 180 min.
Flow rate: 1
Injection volume: 100
Detector: UV 225

CHROMATOGRAM
Retention time: 3

OTHER SUBSTANCES
Simultaneous: adrenocorticotropin hormone and fragments, endorphins, lipotropic hor-
mone and fragments, melanotropin, menotropins, prolactin

REFERENCE
Richter, W.O.; Schwandt, P. Separation of neuropeptides by HPLC: evaluation of different supports,
with analytical and preparative applications to human and porcine neurophysins, [3-lipotropin, ad-
renocorticotropic hormone, and p-endorphin. J.Neurochem., 1985, 44, 1697-1703

SAMPLE
Matrix: solutions
Sample preparation: Prepare a 1 mg/mL solution in 1% ammonium bicarbonate, inject a
100 |xL aliquot.

HPLCVARIABLES
Column: 250 X 4.1 Synchropak RP-P (Synchrom)
Mobile phase: Gradient. A was 0.1% trifluoroacetic acid in water. B was 0.1% trifluoroacetic
acid in n-propanol. A:B from 100:0 to 50:50 over 50 min.
Flow rate: 1
Injection volume: 100
Detector: UV 220

CHROMATOGRAM
Retention time: 41

REFERENCE
Kohr, W. J.; Keck, R.; Harkins, R.N. Characterization of intact and trypsin-digested biosynthetic human
growth hormone by high-pressure liquid chromatography. Anal.Biochem., 1982, 122, 348-359

ANNOTATED BIBLIOGRAPHY
Chang, J.R; Tucker, R.C.; Ghrist, B.R; Coleman, M.R. Non-denaturing assay for the determination of
the potency of recombinant bovine somatotropin by high-performance size-exclusion chromatography.
J.ChromatogrA, 1994, 675, 113-122