## 3 Results and Discussion
### Comparison of Four Rapid N-Glycan Analytical Methods

Table 3: The comparison of the protein sample requirement and testing time between these four rapid methods and conventional 2-AB method. method, respectively. For low abundant glycans, there were minor differences between the 2D-LC-MS method and the rapid 2-AB method. In addition, the cosine value between the 2D-LC-MS method and the rapid 2-AB method for N-glycan data of mAb B was 0.9995, hinting that these two methods offered similar and comparable data. As a relatively new method, the 2D-LC-MS method can provide comparable N-glycan data with less testing time and a lower sample requirement, indicating its great application potential in mAb development. With the advantages of less testing time and a lower sample requirement, these four rapid N-glycan analytical methods would have great application potential in cell line development and will benefit the development of mAbs. Assuming the top 100 cell lines are expanded into shaken culture tubes for simplified fed-batch screening, there are 100 samples for N-glycan analysis. First, the 100 samples must be purified by means of affinity chromatography, and then they must undergo N-glycan analysis via suitable methods. It is worth noting that the purification of mAbs from the medium falls outside the scope of this study, and the comparison of different methods presented in our manuscript is based on the analysis of identical samples--mAbs of high purity. Detailed comparison is listed in Table 1. If using the conventional 2-AB method, the pre-treatment step (glycans release, labelling, etc.) usually requires 2 days, and HPLC analysis usually require 1 h per sample, that is 24 samples per day, which is the limitation step. Therefore, the throughput of the conventional 2-AB method is 24 samples every 3 days, and the glycan analysis of 100 samples require 12.5 days. If using the rapid 2-AB method, 100 sample analysis only requires 6.25 days, saving 6.25 days compared to the conventional 2-AB method. If using the reduction method, 100 sample analysis only requires about 5.26 days, saving about 7.24 days compared to the conventional 2-AB method. If using the off-line IdeS digestion method, 100 sample analysis only requires about 7.69 days, saving about 4.81 days compared to the conventional 2-AB method. When using the 2D-LC-MS method, 100 sample analysis required only about 2.78 days, saving about 9.72 days compared to the conventional 2-AB method. These four rapid methods can significantly reduce the testing time of N-glycan analysis. Furthermore, the 2D-LC-MS method is the most effective at about 78% time saving, which will help to accelerate the cell line development of mAbs. Additionally, the protein sample requirements of these four rapid methods are much lower than that of the conventional 2-AB method. In cell line development, protein materials are one of the limitations for quality analysis due to low titer and small cell culture scale. Usually only lower than milligram-level protein materials are available after affinity chromatography purification. These protein materials need to be allocated for different quality attribute analysis, and the protein materials allocated for N-glycan analysis usually cannot meet the requirement of conventional 2-AB. This is one of the bottlenecks in cell line development. These four rapid methods require only microgram levels of protein samples, especially the 2D-LC-MS method, which needs only 2 microgram protein samples and fits well with the N-glycan analysis in cell line development.