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OBJECTIVE: To establish the system suitability standard for N-glycan profile analysis of monoclonal antibodies. METHODS: LC-MS was used to characterize the N-linked glycoform of the system suitability standard, and the stability was evaluated. The acceptance criteria of system suitability was set according to the method property and the data from method validation. RESULTS: The data of accelerated and long-term stability test indicated that the system suitability standard was stable under storage condition. The N-glycoform of this standard was representative, which covered the main glycoform of monoclonal antibodies. The acceptance criteria of system suitability set for the three analytical METHODS: were as below: the detected chromatogram should be visually similar to representative chromatogram; the resolution between G1F(1, 6) and G1F(1, 3), and G0F percentage should meet the specific requirements; the RSD of G0F retention time should be ≤4%. CONCLUSION: The standard substance for system suitability test and acceptance criteria for three N-glycan profile analytical METHODS are established.
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OBJECTIVE: To validate the HILIC-UPLC method for N-glycan profile analysis of therapeutic antibodies. METHODS: The interlaboratory method validation was performed according to ICH_Q2_R1 guideline and general principles of China Pharmacopoeia (2015 edition) 9101. The validation items included specificity, linearity, accuracy, precision, quantitation limit, range and robustness. RESULTS: The method showed good specificity, accuracy, precision and robustness, and showed a good linearity at a protein range from 100 to 400 μg. The r2 of linear regression equation was above 0.98, and the recoveries were between 86% and 117%. Both the RSDs of peak area percentage and retention time were below 10%, which indicated good precision. The lower quantitation limit of the method was 0.040%, and the range was from 0.040% to 78.751%, which means that single peak at this range could be quantified accurately. Furthermore, robustness evaluation under a series of conditions showed that this method was robust, where the RSD of peak area percentage was below 5% and RSD of retention time was below 1%. CONCLUSION: Interlaboratory validation of HILIC-UPLC provides a methodological verification basis for the improvement of Chinese Pharmacopoeia standards.
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OBJECTIVE: To analyze the N-glycan profile of therapeutic antibodies by UPLC-Qda MS. METHODS: The Nlinked glycan in Fc region was released by PNGase F digestion and labeled by RapiFluor-MS, and labeled glycans were analyzed by UPLC-Qda MS. RESULTS: The UPLC-Qda MS method showed good accuracy in N-glycan quantitaion and qualification, and the Δm/z value of individual N-glycan was in a range of ± 0.5. Three similar biotherapeutic products (SBP) showed a nearly same N-glycan profile with the reference biotherapeutic products (RBP) and the total percentage of fucosylated glycans was comparable, only one fraction of N-glycans had some difference. The major N-glycans of antibodies expressed by CHO cells, NS0 cells and SP2/0 cells were G0F-N, G0F, Man5, G1Fa, G1Fb and G2F, accounting for above 80% of total glycans. Eleven glycoforms were detected in CHO cell expressed antibodies, 22 and 26 glycoforms were detected in NS0 cell and SP2/0 cell expressed antibodies respectively. The N-glycans of NS0 cell and SP2/0 cell expressed antibodies contained more sialylated and galactosylated complex glycoforms, which was related to the antibody half-life in vivo and immunogenicity. CONCLUSION: The HILIC UPLC-Qda MS, as a fast and accurate analytical method, can be used in the quality control of N-glycan profile of antibody.
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ted in a loss of cell fusion,which suggests the 928 site on G2 is crucial for cell fusion and the fusion peptide is likely on G2.