Pavlu B, Gellerfors P
Kabi Pharmacia Peptide Hormones, Stockholm, Sweden.
Bioseparation. 1992;3(5):257-65.
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 used in the study contained two rhGH variants, e.g. LMWGH and Clip I. These variants are structurally very similar to rhGH and exhibited very similar chromatographic behaviour to rhGH, important in evaluation of selectivity. Structural studies revealed that LMWGH had lost the first N-terminal amino acid residue, phenylalanine. Clip I exhibited an increased molecular mass of 32.7 Da for the C-terminal tryptic fragment T18-T19. Temperature and pH were found to influence retention time, sharpness of peaks and selectivity. Furthermore, recoveries were improved from 50% to 99% for rhGH upon the introduction of 0.075% Brij 35, a non-ionic detergent, in the presence of 5% acetonitrile. The optimized HI-HPLC system was found to exhibit good recoveries and excellent selectivity. The system is suitable both as an in-process chromatographic purification step for rhGH, as well as an analytical test method for purity and potency.
一种重组人生长激素制剂（rhGH）已被用于研究温度、pH值和去污剂（Brij 35）浓度对在TSK-苯基-5PW柱上进行疏水相互作用色谱法（HI-HPLC）时的选择性、回收率和保留时间的影响。该研究中使用的rhGH制剂包含两种rhGH变体，即低分子量生长激素（LMWGH）和Clip I。这些变体在结构上与rhGH非常相似，并且表现出与rhGH非常相似的色谱行为，这对于评估选择性很重要。结构研究表明，LMWGH失去了第一个N端氨基酸残基苯丙氨酸。Clip I的C端胰蛋白酶片段T18-T19的分子量增加了32.7 Da。发现温度和pH值会影响保留时间、峰的尖锐度和选择性。此外，在5%乙腈存在的情况下，引入0.075%的非离子去污剂Brij 35后，rhGH的回收率从50%提高到了99%。发现优化后的HI-HPLC系统具有良好的回收率和出色的选择性。该系统既适合作为rhGH的过程中色谱纯化步骤，也适合作为纯度和效价的分析测试方法。
