WO2006051554A1 - A novel process for purification of human growth harmone - Google PatentsSearch our Database of Scientific Publications and Authors. Hydrophobic interaction chromatography of recombinant human growth hormone, Genotropin. Steroids bd PavluP Gellerfors. A recombinant human growth hormone preparation rhGH has been used to study the effects of temperature, pH and detergent Brij 35 concentration on the selectivity, recovery and retention time, during hydrophobic interaction chromatography HI-HPLC on a TSK-phenyl-5PW column. The rhGH preparation hormonee in the study contained two rhGH variants, e.
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The composition and temperature of the mobile phase were optimized for the separation. The method separated eight rhGH variants: Characterization of the purified variants was conducted by liquid chromatography—mass spectrometry tryptic mapping. The novel mobile phase, in combination with the UHPLC system, generated a significantly higher resolution than previously reported reversed-phase LC methods, including pharmacopoeal methods, for analyzing rhGH.
Human growth hormone hGH consists of a single polypeptide chain amino acid residues with two disulfide bridges and a mass of 22 kDa 1.
Modifications of recombinant hGH rhGH may arise during the fermentation and purification processes or during storage of the protein, which can influence the biological activity of the hormone.
Under acidic conditions, the dehydration of normal Asp can yield isoAsp, and both normal and isoAsp rhGH variants have been separated by cation-exchange chromatography, as communicated by a conference poster 3. Methionine residues, at positions 14 and , are prone to oxidation 2 , 4 , 5 , whereas the only remaining methionine Met , located in the interior of the protein 6 , is not oxidized in the native peptide 7.
Reversed-phase liquid chromatography RPLC is used because of its high resolution. It is also the most often used LC technique for analyzing different variants of rhGH. Other techniques, such as capillary LC 9 and non-porous particle 14 methods have also been used. A review of methods for analyzing rhGH variants is provided by Bayol and collaborators Therefore, a need exists for an improved analytical LC method to analyze various rhGH variants.
The potassium borate—acetonitrile mobile phase offered significantly higher selectivity than several other commonly used buffers—mobile phases. The new mobile phase with the UHPLC system resulted in significantly higher resolution than in previously described methods.
Characterization of the previously purified rhGH variants was performed by liquid chromatography—mass spectrometry LC—MS tryptic mapping date. Acetonitrile LiChrosolv , potassium phosphate, boric acid and affinity purified trypsin catalogue number were from Merck Darmstadt, Germany , and 1-propanol Chromasolv , tris hydroxymethyl aminomethane Tris and trifluoroacetic acid TFA were from Sigma-Aldrich St.
Ammonium bicarbonate was from Fluka Buchs, Switzerland. The production, purification and characterization of the rhGH variants were previously performed at Pfizer, as summarized in the following.
The desPhe1 rhGH was obtained from the production process for rhGH Pfizer , from a side fraction at an early chromatographic step during the purification procedure. The clip ThrTyr variant was from a side fraction that eluted at an early step in the rhGH purification process, which was further purified by the phenyl HIC column Characterization was performed by LC—MS tryptic mapping and additional analytical techniques.
The previously performed characterization was briefly repeated in the present work to obtain confirmation. The rhGH forms were analyzed, both separately and spiked to a native rhGH laboratory sample at a concentration of 0. The flow rate was 0. Potassium phosphate, pH 7. In addition, a method that was essentially described by the United States Pharmacopeia USP and the European Pharmacopoeia EP 21 , 22 was used during the method development, using a mobile phase consisting of Because of the increased backpressure, the flow rate was reduced to 0.
The organic percentage in the mobile phases was slightly adjusted to obtain approximately the same retention factors. Characterizations of the purified rhGH variants were conducted to confirm the previous characterizations.
The mobile phase A was 0. The QTOF was set as follows: Spectra were recorded from 50—2, scan time 0. Thus, the method is sensitive to small temperature fluctuations and accurate and reliable control of the column oven temperature is necessary.
For most analyses, a C18 column was used with a mobile phase consisting of 25 mM potassium borate pH 8. The flow rate and detection was 0. The variants are indicated in the figure based on individual injections as follows: The largest peak in the chromatograms is the rhGH native form. Further details are provided in the paper. Isocratic elution has been shown to be the most effective method for separating rhGH variants by RPLC, and this has been used in the pharmacopoeal methods USP and EP and in many reports 4 , 5 , 8 , 14 , 19 , The use of a mobile phase with propanol typically 1-propanol as the organic modifier, combined with Tris buffers, have been used in USP and EP methods 21 , 22 and by several authors 4 , 8 , 23 , and propanol—phosphate 5 , 14 , 19 and acetonitrile—phosphate 2 , 5 , 12 have also been used.
In developing the present method, several other buffer components and various pHs were tested, in addition to gradient elution; however, these were not successful.
For example, the mono-deamidated rhGH and the desPhe1 variants coeluted with the primary rhGH peak in both methods. The retention times for the rhGH variant samples 0. Accuracy was calculated from the difference between the observed values for the spiked rhGH laboratory samples with 1. These results indicate acceptable intermediate precision, linearity and accuracy at low levels of rhGH variants when analyzed by the optimized method.
The detection limit was not rigorously investigated; however, distinct increased peaks, compared with the unspiked rhGH laboratory sample, were observed when the sample was spiked with 0. The selectivity of the optimized method. The following samples were analyzed separately: In the analysis of rhGH described in this study, a C18 column with a mobile phase of pH 8.
A variety of mobile phase pH ranges was initially evaluated before settling on pH 8. The use of a mobile phase at pH 8. Both 1-propanol and acetonitrile were separately tested as organic modifiers in the mobile phase, with acetonitrile demonstrating the best selectivity and resolution.
Acetonitrile is usually the preferred organic phase in LC, based on its physical properties such as low viscosity, which offers a high diffusion rate and higher resolution, especially for high molecular mass compounds such as proteins.
In contrast, propanol, with a significantly higher viscosity, results in more peak-broadening. This allows for detection by absorbance at nm, which gives a high response and a low baseline noise. Borate and phosphate have the lowest UV cutoffs of the most commonly used LC buffers, which is why these buffers were the focus of this evaluation. Canova-Davis and collaborators found that 1-propanol was more effective than acetonitrile for the RPLC separation of Met14 sulfoxide rhGH and native rhGH, although acetonitrile was more effective for separating Met14 and Met sulfoxide variants of rhGH when using a polymer column with a phosphate buffer mobile phase 5.
The differences observed in the results of this study and previous results 5 can be explained by several factors, including differences in the column packing material, buffer components and pH. The oxidized T11 fragment eluted in two distinct peaks at The T2 peak was not completely resolved from the previous peak and there were some minor disturbances in the tryptic map chromatograms; however, this did not cause any problems in the QTOF-MS analysis.
Because Asn and Asn are the only possible candidates for deamidation in T15, these amino acids must have been deamidated to aspartic acid or to isoaspartic acid 2 , 3. The rhGH trisulfide sample gave a TT21 complex mass of Da, corresponding to an increase of 32 Da, whereas the mass of the other disulfide complex T6-T16 was unchanged, compared with the reference.
An increase in the mass of 32 Da could also indicate two oxidations. However, the TT21 fragment complex does not contain any easily oxidized methionine residues, and if the cysteine residues were oxidized, the retention time of the complex would decrease due to the decreased hydrophobicity. Trisulfide complexes of tryptic peptides from an antibody, when compared with the corresponding disulfide complexes, also showed increased hydrophobicity and increased retention time when analyzed by RPLC The dipeptide QT from the clipped T14 was not found and may have eluted in the void peak.
The LC—MS results of analysis for the intact rhGH variants and tryptic fragments demonstrated the expected mass differences, relative to the reference rhGH native form, confirming the results of the more detailed previous characterization. Chromatography was performed on a C18 column, eluted with an acetonitrile gradient with TFA as the additive: The rhGH reference peptide maps are shown as mirror images and the most characteristic tryptic fragments are indicated in the figures.
A disturbance in the baseline occurs at approximately 8. The method was the result of method development experiments that explored the effects of temperature, mobile phase buffers and organic modifiers. The newly developed mobile phase resulted in much improved selectivity, and when combined with the UHPLC system, produced a significantly higher resolution for separating rhGH variants than previously reported RPLC methods, including pharmacopoeal methods.
Characterization of the purified variants was performed by LC—MS tryptic mapping. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Close mobile search navigation Article navigation.
View large Download slide. Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone. Separation and identification of growth hormone variants with high performance liquid chromatography techniques. Sensitive asparagine and aspartic acid positions in biosynthetic human growth hormone bhGH: Deamidation and isomerization in neutral solutions; Poster No. Isolation and characterization of a sulfoxide and a desamido derivative of biosynthetic human growth hormone.
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