O'Connor J V
Genentech, Inc., South San Francisco, CA 94080, USA.
Dev Biol Stand. 1999;97:39-47.
Variants of intact polypeptides/proteins ranging in mass from 6,500 to 70,000 Da were easily separated using reversed-phaseHPLC (rpHPLC) or affinity chromatography. A variant of rhlGF-I, where the racemization of a serine residue was detected in the intact molecule, was resolved from rhlGF-I within 25 minutes by rpHPLC. Other variants of rhlGF-I separated by this method include methionine sulphoxide at position 59, des Gly1, des Gly1Pro2, Glu for Asp substitution at position 20 and incorrectly folded IGF-I. For rhDNase (approximately 40 kDa), affinity chromatography was able to clearly resolve three different amino acids (Asn, Asp and iso-Asp) at position 74 of the intact glycoprotein. The presence or absence of O-linked sugars on Thr -37 of recombinant human thrombopoietin was rapidly demonstrated by rpHPLC. While the separation of these types of variants is essential, the demonstration of biological activity is critical for designing specifications that allow the administration of these proteins into humans. Once a correlation exists between the variant and its biological activity, control of the manufacturing process can be better achieved with analytical methodology.
质量范围在6500至70000道尔顿的完整多肽/蛋白质变体，使用反相高效液相色谱（rpHPLC）或亲和色谱法很容易分离。在完整分子中检测到丝氨酸残基消旋化的重组人胰岛素样生长因子-I（rhlGF-I）变体，通过rpHPLC在25分钟内与rhlGF-I分离。用这种方法分离的rhlGF-I的其他变体包括59位的甲硫氨酸亚砜、去甘氨酸1、去甘氨酸1脯氨酸2、20位天冬氨酸被谷氨酸取代以及折叠错误的胰岛素样生长因子-I。对于重组人脱氧核糖核酸酶（rhDNase，约40 kDa），亲和色谱法能够清晰地分离完整糖蛋白74位的三种不同氨基酸（天冬酰胺、天冬氨酸和异天冬氨酸）。通过rpHPLC迅速证实了重组人血小板生成素37位苏氨酸上O-连接糖的存在与否。虽然分离这些类型的变体至关重要，但证明其生物活性对于制定允许将这些蛋白质施用于人体的规范至关重要。一旦变体与其生物活性之间存在关联，就可以通过分析方法更好地控制制造过程。
