## 3 Results and Discussion
### Comparison of Four Rapid N-Glycan Analytical Methods  
Based on the above data, we compared the six major glycans of mAb A from these four methods in Figure 4. [PERSON]'s multiple comparison test was widely used in comparisons of different groups [29; 30] and was performed in this study to compare the differences between any two methods. The adjusted P values between any two methods were 0.9999 or >0.9999, which meant that there were no significant differences between any two methods. For highly abundant glycans, such as G0F, G1F, and G2F, these four methods provided comparable data, despite some minor differences for low abundant glycans, such as G0F-GN, G1F-GN, and Man5. It was speculated that these minor differences were due to the dilution in the 2D-LC-MS system, the efficiency of pre-treatment, and response differences between MS and fluorescence detectors. The comparison of these four rapid methods is listed in Table 2. The rapid 2-AB method analyzed N-glycans using a fluorescence detector at the released N-glycan level, which differed from the other three methods using an MS detector at the subunit level. The N-glycans release step, labelling step, subsequent purification step, and fluorescence detection could influence the N-glycan data. For the reduction method, the off-line IdeS digestion method, and the 2D-LC-MS method, the reduction step, off-line IdeS digestion step, and on-line IdeS digestion step could influence the data of N-glycans. The 2D-LC-MS method utilized on-line immobilized IdeS columns for digestion and RP columns for on-line desalting. It had an on-line dilution effect, which may induce a variation in the method. Cosine similarity, which is one of the most widely used approaches for a similarity calculation, was used to further compare the similarity of N-glycan data from these four rapid methods [31]. The smaller the angle of the two non-zero vectors a and b, the closer the cosine value is to 1, and the more their directions coincide, the more similar they are. The cosine value of six major glycans between the 2D-LC-MS method and the rapid 2-AB method was 0.9853, the cosine value of six major glycans between the 2D-LC-MS method and the reduction method was 0.9895, and the cosine value of six major glycans between the 2D-LC-MS method and the off-line IdeS digestion method was 0.9946. Thismeant N-glycan data from the 2D-LC-MS method and other methods were similar and comparable. Detailed information is shown in Supplementary Materials, Table S4. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Method** & **Analytical Principle** & **Pre-Treatment** & **Column** & **Detector** \\ \hline \multirow{4}{*}{Rapid 2-AB method} & N-glycans is released from the & Denaturation, & & \\  & antibody by PNGase F, labelled & N-glycan release, & & \\  & by 2-AB, and tested by a & labelling and & & \\  & fluorescence detector. & purification. & & \\  & The antibody is reduced to & & & \\  & generate HC and analyzed & Off-line DTT & & \\  & N-glycans by MS at the subunit & reduction. & RP column & Q-TOF MS detector \\  & level. & & & \\  & The antibody is off-line digested & & & \\   Off-line IdeS digestion & by IdeS to generate Fc/2 and & & & \\   method & analyzed N-glycans by MS at the & & & \\  & subunit level. & & & \\  & The antibody is on-line digested & & & \\  & by the column with immobilized & & & \\  2D-LC-MS method & IdeS to generate Fc/2 and & & & \\  & analyzed N-glycans by MS at the & & & \\  & subunit level. & & & \\  & & & & \\ \end{tabular}
\end{table} 
Table 2: The comparison of these four rapid methods. Figure 4: Comparison of six major glycans of mAb A from the reduction method (n = 3), the off-line IdeS digestion method (n = 3), the 2D-LC-MS method with on-line immobilized IdeS digestion (n = 3), and the rapid 2-AB method (n = 3). Furthermore, we compared the protein sample requirement and testing time between these four methods and the conventional 2-AB method. Details are listed in Table 3. The 2-AB method is considered the gold standard for N-glycan analysis.