**4. Conclusions**

The analytical characterization of biopharmaceutical is still challenging for biotech industry to meet the requirements. Conventional methods, such as chromatography and electrophoresis, are routinely used because they are easy to use, robust, and, cost effective. Current trends for characterization are in-depth and well characterized. Current advances in instrumentation can help to follow those trends and characterize very complex heterogeneity from various PTMs. MS is the most powerful instrument among them, which provides high resolution, accurate, and confident data with rich information from primary structure (intact mass and peptide mapping) to high order structures (PTMs and HDX).

In this chapter, several workflows are summarized for intact mass determination, primary structure analysis, and determination and quantitation of various PTMs using chromatography with online detection by MS. Those conventional approaches were assessed by the current MAM approaches primarily by peptide mapping analysis using MS.

MAM approach has been introduced, which is able to identify and quantify several attributes at once. In this chapter, glycosylation, deamidation/isomerization, C-terminal Lys variants, and N-terminal cyclization are investigated by using MAM approach, and the performance was compared to the conventional methods such as HILIC oligosaccharide analysis and CEX charge variant analysis. The results confirmed that MAM approach is quite comparable for those from conventional independent approaches.

In this chapter, we showed that MAM approach for biopharmaceutical characterization is quite comparable for typical conventional approaches using HILIC and CEX. This result conveys that MAM workflow can be extended to other related area of biopharmaceutical development as follows. MAM approach may help to select best cell lines for producing biopharmaceuticals, to support process control for upstream and downstream, and monitor critical attributes for production. MAM approach will also gain attention for the development of biosimilar requiring in-depth structural analysis for similarity.

## **Acknowledgements**

This research was partially supported by Industry-Academy Cooperation Program of SMIT funded by BIOnSYSTEMS Ltd., Republic of Korea. Woojeong Kim was supported by the National Research Foundation of Korea (NRF) and the Center for Women In Science, Engineering and Technology (WISET) Grant funded by the Ministry of Science and ICT (MSIT) under the Program for Returners into R&D, Republic of Korea.

## **Conflict of interest**

result in mass shifts compared to those intact peptides, and this gives clues for detecting PTMs by considering the mass differences. Most of those PTMs may not be separated from their unmodified form by conventional approaches. For those cases, MAM approach is a pos-

**Peak N-terminal structure of light chains Relative content (%)**

**Table 8.** The relative contents of N-terminal cyclization variant from rituximab identified by CEX analysis.

**(Da)**

**Table 9.** The relative contents of N-terminal cyclization variant from rituximab identified by MAM analysis.

L:T1 1823.9993 1823.9949 −2.41 0.93 ± 0.01

L:T1 pyroGlu 1806.9727 1806.9731 0.22 99.08 ± 0.01

**Mass (Da) Error** 

**(ppm)**

**Relative content (%)**

Acidic pyroGlu/pyroGlu 5.26 ± 0.01 M pyroGlu/pyroGlu 89.40 ± 0.12 B1 pyroGlu/pyroGlu 3.42 ± 0.08 B2 Gln/pyroGlu 1.92 ± 0.05

**Peptide Number Change Calculated mass** 

L: light chain, T: tryptic peptide, and pE: pyro-glutamate.

M: major and B: basic.

32 Biopharmaceuticals

QIVLSQSPAI LSASPGEK

pEIVLSQSPAI LSASPGEK

sible alternative for quantifying those PTMs.

**Figure 17.** Profiles of N-terminal cyclization variants for rituximab.

The authors have nothing to disclose.
