Validation of a Peptide Map for Recombinant Porcine Growth Hormone and Application to Stability Assessment

Susan A. Charman, Louise E. McCrossin, William N. Charman

Research output: Contribution to journalArticleResearchpeer-review

Abstract

A reverse-phase HPLC method for the analysis of tryptic digests of recombinant porcine growth hormone (pGH) has been developed and validated. Digestion was performed at 4°C for a 20-hr period with TPCK-treated trypsin at a 1:20 (w/w) trypsin:pGH ratio. Gradient elution HPLC, using an Aquapore RP300 C8 column, was incorporated for separation of the digestion products and peak identification was carried out by mass spectrometry (MS). The digestion procedure and subsequent chromatography were linear in the initial concentration range of 4.55 – 45.46 µM (100 to 1000 µg/mL) pGH. The variability in the fragment retention times was low and the normalized peak area variability was less than 5% for all but three of the fragments. The utility of the trypsin digestion and chromatography procedures has been demonstrated by assessing chemical changes in pGH induced by incubation at elevated pH. Upon incubation of pGH in 0.2 M Tris buffer at pH 9 (ionic strength adjusted to 0.5 with NaCl) and 37°C over a period of 400 hr, significant degradation in the regions corresponding to the digestion fragments T23–T25 (residues 181–182 linked by a disulfide bond to residues 184–191), T9 (residues 96–108), and T5–T18 (residues 43– 64 linked by a disulfide bond to residues 158–166) was observed. The disappearance of the peaks corresponding to fragments T23–T25 and T9 both displayed apparent first-order degradation kinetics over the time period investigated with half-lives of 131 and 154 hr, respectively. The disappearance of the peak corresponding to fragment T5–T18 was complicated by poor resolution of the parent peak and the degradation products. These results demonstrate the utility and limitations of the mapping procedure for the determination of the reaction kinetics for pGH.

Original languageEnglish
Pages (from-to)1471-1477
Number of pages7
JournalPharmaceutical Research
Volume10
Issue number10
DOIs
Publication statusPublished - 1993
Externally publishedYes

Keywords

  • peptide map
  • porcine growth hormone
  • protein stability

Cite this

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title = "Validation of a Peptide Map for Recombinant Porcine Growth Hormone and Application to Stability Assessment",
abstract = "A reverse-phase HPLC method for the analysis of tryptic digests of recombinant porcine growth hormone (pGH) has been developed and validated. Digestion was performed at 4°C for a 20-hr period with TPCK-treated trypsin at a 1:20 (w/w) trypsin:pGH ratio. Gradient elution HPLC, using an Aquapore RP300 C8 column, was incorporated for separation of the digestion products and peak identification was carried out by mass spectrometry (MS). The digestion procedure and subsequent chromatography were linear in the initial concentration range of 4.55 – 45.46 µM (100 to 1000 µg/mL) pGH. The variability in the fragment retention times was low and the normalized peak area variability was less than 5{\%} for all but three of the fragments. The utility of the trypsin digestion and chromatography procedures has been demonstrated by assessing chemical changes in pGH induced by incubation at elevated pH. Upon incubation of pGH in 0.2 M Tris buffer at pH 9 (ionic strength adjusted to 0.5 with NaCl) and 37°C over a period of 400 hr, significant degradation in the regions corresponding to the digestion fragments T23–T25 (residues 181–182 linked by a disulfide bond to residues 184–191), T9 (residues 96–108), and T5–T18 (residues 43– 64 linked by a disulfide bond to residues 158–166) was observed. The disappearance of the peaks corresponding to fragments T23–T25 and T9 both displayed apparent first-order degradation kinetics over the time period investigated with half-lives of 131 and 154 hr, respectively. The disappearance of the peak corresponding to fragment T5–T18 was complicated by poor resolution of the parent peak and the degradation products. These results demonstrate the utility and limitations of the mapping procedure for the determination of the reaction kinetics for pGH.",
keywords = "peptide map, porcine growth hormone, protein stability",
author = "Charman, {Susan A.} and McCrossin, {Louise E.} and Charman, {William N.}",
year = "1993",
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language = "English",
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journal = "Pharmaceutical Research",
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Validation of a Peptide Map for Recombinant Porcine Growth Hormone and Application to Stability Assessment. / Charman, Susan A.; McCrossin, Louise E.; Charman, William N.

In: Pharmaceutical Research, Vol. 10, No. 10, 1993, p. 1471-1477.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Validation of a Peptide Map for Recombinant Porcine Growth Hormone and Application to Stability Assessment

AU - Charman, Susan A.

AU - McCrossin, Louise E.

AU - Charman, William N.

PY - 1993

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N2 - A reverse-phase HPLC method for the analysis of tryptic digests of recombinant porcine growth hormone (pGH) has been developed and validated. Digestion was performed at 4°C for a 20-hr period with TPCK-treated trypsin at a 1:20 (w/w) trypsin:pGH ratio. Gradient elution HPLC, using an Aquapore RP300 C8 column, was incorporated for separation of the digestion products and peak identification was carried out by mass spectrometry (MS). The digestion procedure and subsequent chromatography were linear in the initial concentration range of 4.55 – 45.46 µM (100 to 1000 µg/mL) pGH. The variability in the fragment retention times was low and the normalized peak area variability was less than 5% for all but three of the fragments. The utility of the trypsin digestion and chromatography procedures has been demonstrated by assessing chemical changes in pGH induced by incubation at elevated pH. Upon incubation of pGH in 0.2 M Tris buffer at pH 9 (ionic strength adjusted to 0.5 with NaCl) and 37°C over a period of 400 hr, significant degradation in the regions corresponding to the digestion fragments T23–T25 (residues 181–182 linked by a disulfide bond to residues 184–191), T9 (residues 96–108), and T5–T18 (residues 43– 64 linked by a disulfide bond to residues 158–166) was observed. The disappearance of the peaks corresponding to fragments T23–T25 and T9 both displayed apparent first-order degradation kinetics over the time period investigated with half-lives of 131 and 154 hr, respectively. The disappearance of the peak corresponding to fragment T5–T18 was complicated by poor resolution of the parent peak and the degradation products. These results demonstrate the utility and limitations of the mapping procedure for the determination of the reaction kinetics for pGH.

AB - A reverse-phase HPLC method for the analysis of tryptic digests of recombinant porcine growth hormone (pGH) has been developed and validated. Digestion was performed at 4°C for a 20-hr period with TPCK-treated trypsin at a 1:20 (w/w) trypsin:pGH ratio. Gradient elution HPLC, using an Aquapore RP300 C8 column, was incorporated for separation of the digestion products and peak identification was carried out by mass spectrometry (MS). The digestion procedure and subsequent chromatography were linear in the initial concentration range of 4.55 – 45.46 µM (100 to 1000 µg/mL) pGH. The variability in the fragment retention times was low and the normalized peak area variability was less than 5% for all but three of the fragments. The utility of the trypsin digestion and chromatography procedures has been demonstrated by assessing chemical changes in pGH induced by incubation at elevated pH. Upon incubation of pGH in 0.2 M Tris buffer at pH 9 (ionic strength adjusted to 0.5 with NaCl) and 37°C over a period of 400 hr, significant degradation in the regions corresponding to the digestion fragments T23–T25 (residues 181–182 linked by a disulfide bond to residues 184–191), T9 (residues 96–108), and T5–T18 (residues 43– 64 linked by a disulfide bond to residues 158–166) was observed. The disappearance of the peaks corresponding to fragments T23–T25 and T9 both displayed apparent first-order degradation kinetics over the time period investigated with half-lives of 131 and 154 hr, respectively. The disappearance of the peak corresponding to fragment T5–T18 was complicated by poor resolution of the parent peak and the degradation products. These results demonstrate the utility and limitations of the mapping procedure for the determination of the reaction kinetics for pGH.

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KW - protein stability

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JO - Pharmaceutical Research

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