| 1. |
- Arnell, Robert, et al.
(författare)
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Biotechnological Approach to the Synthesis of 9α-Hydroxylated Steroids
- 2007
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Ingår i: Preparative Biochemistry & Biotechnology. - : Informa UK Limited. - 1082-6068 .- 1532-2297. ; 37:4, s. 309-321
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Tidskriftsartikel (refereegranskat)abstract
- The steroid 9α-hydroxylase gene has been cloned from Mycobacterium smegmatis into Escherichia coli BL21. Progesterone added to bioreactors was subjected to in vivo transformation into 9α-hydroxyprogesterone. In 7 days, 43.6 mg9α-hydroxyprogesterone was formed from 53.8 mg/L progesterone. The enzyme also has shown evidence of processing 4-androstene-3,17-dione in vivo. An extensive analytical method development, including LLE, HPLC-DAD, MS, andNMR was performed to verify the product and to enable a quantitative analysis. Protocols for analytical and preparative separation have been developed, using binaphtol as internal standard. Both the growth pattern and the bioconversion ratewere unaffected by the presence of binaphtol in the bioreactor. The enzyme was purified by immobilised metal affinity and ion exchange chromatography, resulting in low in vitro activity.
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| 3. |
- Lindholm, Johan, 1974-
(författare)
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Development and Validation of HPLC Methods for Analytical and Preparative Purposes
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis concerns the development and validation of high performance liquid chromatography (HPLC) methods aimed for two industrially important areas: (i) analysis of biotechnological synthesis and (ii) determination of adsorption isotherm parameters. There is today a lack of detailed recommendations for analytical procedures in the field of biotechnological production of drugs. Therefore, guidelines were given for analytical development and validation in this field; the production of 9α-hydroxyprogesterone was used as model. In addition, a rapid method using HPLC coupled with diode-array-detection (DAD) and mass spectrometry (MS), was developed for the preliminary identification and quantification of the product. In addition, requirements and recommendations were developed for the selection of the internal standard and for its inclusion in the process liquid. By using this approach the precision and accuracy of the quantitative method were considerably improved. Preparative chromatography is a powerful separation method for the purification of pure compounds from more or less complex sample mixtures. One such mixture can be the process liquid from a fermentation, another example can be a racemic mixture of compounds whose enantiomeric constituents must be isolated. Computer-assisted modeling can be used to optimize preparative chromatography. However, competitive adsorption isotherm parameters are required as input data for the computer simulations. In this thesis, a new injection technique, based on a firm theoretical basis, was developed for the peak perturbation (PP) method allowing the determination of binary competitive adsorption isotherm parameters from a broad concentration range. With the new method the determination of adsorption isotherm parameters from a quaternary mixture could be done for the first time. The profiles simulated with these parameters showed excellent agreement with the corresponding experimental profiles, validating the accuracy of the adsorption isotherm parameters derived by the new method.
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| 6. |
- Lindholm, Johan, et al.
(författare)
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Investigation of the adsorption behaviour of a chiral model compound on a tartardiamide-based network-polymeric chiral stationary phase
- 2005
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Ingår i: Journal of Chromatography A. ; 1095:1-2, s. 50-59
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Tidskriftsartikel (refereegranskat)abstract
- The adsorption behaviour of the enantiomers of 2-phenylbutyric acid on the chiral stationary phase (CSP) Kromasil CHI-TBB was studied using hexane/MTBE (90/10) as eluent. Adsorption isotherms were acquired at 40 different enantiomer concentrations in the interval between 7.6 μM and 305 mM, an approximately 40,000-fold dynamic range. The adsorption data fitted well to the bi-Langmuir model, indicating a heterogeneous surface with two different types of adsorption sites having different equilibrium constants and capacities; namely one chiral site and one non-chiral site. A comparison with earlier adsorption studies on modern CSPs revealed that the capacity value of the “true” chiral site of Kromasil CHI-TBB is the largest reported so far. The elution profiles simulated with these parameters show excellent agreement with the corresponding experimental profiles. Guidelines for comparisons of loading capacities of CSPs are presented.
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| 7. |
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| 8. |
- Lindholm, Johan, et al.
(författare)
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Use of liquid chromatography-diode-array detection and mass spectrometry for rapid product identification in biotechnological synthesis of a hydroxyprogesterone
- 2003
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Ingår i: Journal of Chromatography A. - 0021-9673 .- 1873-3778. ; 992:1-2, s. 85-100
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Tidskriftsartikel (refereegranskat)abstract
- In exploratory scale biotechnological process development, the product must be rapidly identified although a reference compound may not always be available. LC-diode-array detection and MS were used for this purpose in a process producing 9alpha-hydroxyprogesterone from progesterone as substrate. The electrospray ionization mass spectrometer was combined with an ion trap mass spectrometer for the second generation MS. The preliminary identification, which could be carried out within the course of a day, confirmed that the product was a hydroxyprogesterone. The final identification step, which was much more material intensive and hence time consuming, involved a two-step preparative separation to yield quantities necessary for definitive product identification based on 1H- and 13C NMR.
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