## 3 Results and Discussion
### Comparison of Four Rapid N-Glycan Analytical Methods
The greatest disadvantages of the 2-AB method is a long and laborious procedure with overnight enzymatic incubations, multi-hour labelling reactions, and purification steps, usually several days for the whole testing [32]. For the rapid 2-AB method used in this study, the testing time was only 4 h with the rapid N-glycans release and subsequent rapid purification steps. The reduction method tests N-glycan at the subunit level, which can minimize the complexity caused by glycan pairs at the intact protein level [6]. In this study, DTT was used as the reducing reagent to cleave the disulfide bond between the antibody chains into HC and LC for subsequent N-glycan analysis using LC-MS. This method required only 74 min per sample, significantly shortening the testing time compared to the conventional 2-AB method. The off-line IdeS digestion method also tested N-glycan at the subunit level and had advantages over other methods at the intact protein level. The difference was that it used the IdeS to enzymatically digest antibodies into F(ab)2 and Fc/2 [22] and analyzed N-glycans of Fc/2 using LC-MS. This method had a similar testing time to the reduction method; 104 min per sample in this study. The 2D-LC-MS method shared the same principle with the off-line IdeS digestion method, just changing off-line digestion to on-line digestion, which improved analytical efficiency and reduced testing time. Based on the rapid on-line digestion hyphenated MS detection in this study, this method took only 40 min per sample in this study. In addition to being time saving, these four rapid methods have a smaller sample requirement than that of the conventional 2-AB method, which can reduce the sample requirement from milligrams to micrograms. A milligram-level protein sample is necessary for the conventional 2-AB method due to the limitations of purification steps. The sample requirement of these four rapid methods is 40 \(\upmu\)g for the rapid 2-AB method, 50 \(\upmu\)g for the reduction method, 50 \(\upmu\)g for the off-line IdeS digestion method, and only 2 \(\upmu\)g for the 2D-LC-MS method. Among the four rapid methods, the 2D-LC-MS method is superior to the conventional 2-AB method due to a lower protein sample requirement. Since the 2D-LC-MS method showed the best performance in terms of the testing time and protein sample requirement, we further evaluated the quantitative performance of the 2D-LC-MS method using mAb B by comparing the 2D-LC-MS method and the rapid 2-AB method. Detailed information is shown in Supplementary Materials, Figure S2 and Table S5. G0F was the prominent glycan of mAb B, the ratio from the 2D-LC-MS method was 82.13%, and the ratio from the rapid 2-AB method was 82.94%, suggesting highly similar results. The ratios of G1F were 11.58% and 12.22% from the 2D-LC-MS method and the rapid 2-AB \begin{table}
\begin{tabular}{p{113.8 pt} p{113.8 pt} p{113.8 pt} p{113.8 pt} p{113.8 pt} p{113.8 pt}} \hline \hline \multirow{2}{*}{**Method**} & **Protein Sample Requirement per Analysis** & **Total Testing Time per Analysis** & **Total Testing Time per 100 Samples** & **Time for 100 Samples** & **Time Saving Ratio for 100 Samples** \\ \hline
**Conventional 2-AB method** & 1 mg [33] & 1–3 days [17] & 24 samples per 3 days * & 12.5 days & NA & NA \\
**Rapid 2-AB method** & 40 \(\upmu\)g & 4 h & 16 samples per day \# & 6.25 days & 6.25 days & 50\% \\
**Reduction method** & 50 \(\upmu\)g & 74 min & \(\sim\)19 samples per day & \(\sim\)5.26 days & \(\sim\)7.24 days & \(\sim\)58\% \\
**Off-line IdeS digestion method** & 50 \(\upmu\)g & 104 min & \(\sim\)13 samples per day & \(\sim\)7.69 days & \(\sim\)4.81 days & \(\sim\)38\% \\
**2D-LC-MS method** & 2 \(\upmu\)g & 40 min & 36 samples per day & \(\sim\)2.78 days & \(\sim\)9.72 days & \(\sim\)78\% \\ \hline \hline \end{tabular} Note.* * based on 2 days for pretreatment of 24 samples and 60 min per sample for HPLC testing. # based on 8 h for pretreatment of 16 samples and 60 min per sample for HPLC testing.  
\end{table}