Characterization of maleuric acid derivatives on transgenic human monoclonal antibody due to post-secretional modifications in goat milk.
A fully human antibody to tumor necrosis factor-alpha was expressed in the mammary glands of transgenic goats. The goat expressed antibody (gAb) is heterogeneous and has several isoforms due to typical cellular post-translational modifications. In addition, one post-secretional modification on gAb was discovered by high-resolution cation exchange chromatography (CIEX). The presence of these variants in the final product was shown to be dependent upon the initial milk storage and traditional purification methodologies used. These observations allow for the development of new sample recovery and purification processes to eliminate these variants. Various enzymatic treatments were used to characterize different gAb heavy chain C-terminal lysine and sialic acid variants. In addition, an unknown derivative with the additional mass of 140 Da was found in transgenic gAb using mass spectrometry (MS). The modification sites were identified as the N-termini of gAb light chains and heavy chains using Q-TOF MS. Characterization of transgenic gAb isoforms was facilitated by utilizing different enzymes, CIEX and MS techniques. A maleuric acid modification on the N-terminal portion of gAb was shown to be consistent with the available data characterizing this new derivative of transgenic gAb isoforms in goat milk.
Santora
L C
LC
Abbott Bioresearch Center, 100 Research Drive, Worcester, MA 01605, USA. Ling.Santora@abbott.com
Stanley
K
K
Krull
I S
IS
Grant
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D006041:Goats; D006801:Humans; D008298:Maleates / Q000737:chemistry*; D013058:Mass Spectrometry; D008892:Milk / Q000737:chemistry*; D019076:Transgenes
D000911:Antibodies, Monoclonal; D008298:Maleates