Liu Renpeng, Giddens John, McClung Colleen M, Magnelli Paula E, Wang Lai-Xi, Guthrie Ellen P
a New England Biolabs Inc. , Ipswich , MA 01938.
b Department of Chemistry & Biochemistry ; University of Maryland , College Park , MD 20742.
MAbs. 2016;8(2):340-6. doi: 10.1080/19420862.2015.1113361. Epub 2015 Oct 29.
Glycosylation affects the efficacy, safety and pharmacokinetics/pharmacodynamics properties of therapeutic monoclonal antibodies (mAbs), and glycoengineering is now being used to produce mAbs with improved efficacy. In this work, a glycoengineered version of rituximab was produced by chemoenzymatic modification to generate human-like N-glycosylation with α 2,6 linked sialic acid. This modified rituximab was comprehensively characterized by liquid chromatography-mass spectrometry and compared to commercially available rituximab. As anticipated, the majority of N-glycans were converted to α 2,6 linked sialic acid, in contrast to CHO-produced rituximab, which only contains α 2,3 linked sialic acid. Typical posttranslational modifications, such as pyro-glutamic acid formation at the N-terminus, oxidation at methionine, deamidation at asparagine, and disulfide linkages were also characterized in both the commercial and glycoengineered mAbs using multiple enzymatic digestion and mass spectrometric analysis. The comparative study reveals that the glycoengineering approach does not cause any additional posttranslational modifications in the antibody except the specific transformation of the glycoforms, demonstrating the mildness and efficiency of the chemoenzymatic approach for glycoengineering of therapeutic antibodies.
糖基化会影响治疗性单克隆抗体（mAb）的疗效、安全性以及药代动力学/药效学特性，目前糖基工程正被用于生产疗效更佳的单克隆抗体。在本研究中，通过化学酶法修饰制备了一种糖基工程化的利妥昔单抗，以生成具有α 2,6连接唾液酸的类人N-糖基化。通过液相色谱-质谱联用对这种修饰后的利妥昔单抗进行了全面表征，并与市售利妥昔单抗进行了比较。正如预期的那样，与仅含有α 2,3连接唾液酸的CHO细胞生产的利妥昔单抗相比，大多数N-聚糖都转化为α 2,6连接的唾液酸。还使用多种酶切和质谱分析对市售和糖基工程化单克隆抗体中的典型翻译后修饰进行了表征，如N端焦谷氨酸的形成、甲硫氨酸的氧化、天冬酰胺的脱酰胺以及二硫键连接。比较研究表明，糖基工程方法除了导致糖型的特异性转变外，不会在抗体中引起任何额外的翻译后修饰，这证明了化学酶法在治疗性抗体糖基工程中的温和性和高效性。
