**5. Glycosylation profiling of a variety of monoclonal antibodies**

IgG1 is the most abundant antibody in human body. Other antibodies are IgG2, IgG3, and IgG4 (**Figure 12**). Generally, IgG1, IgG3, and to some extent, IgG4 are formed against protein antigens. IgG2 is the major subclass formed against repetitive T cell-independent polysaccharide structures found on encapsulated bacteria [22]. Most of the biotherapeutics predominantly belong to IgG1 subclass. The antibodies from subclasses other than IgG1, as well as numerous engineered forms, are also gaining plenty of interest for use as biotherapeutics (**Figure 12**).

Several commercially available IgGs were recently analyzed in-house using TSKgel FcR-IIIA-NPR column to compare their elution profiles. Almost all IgGs yielded the typical 3-peak separation profile although there was substantial difference in each peak height between IgGs. Generally, IgG1, IgG3 and IgG4 subclasses bind to TSKgel FcR-IIIA-NPR whereas IgG2 does not have affinity. Due to

#### **Figure 12.**

*A structural representation of the IgG subclasses and the variation within these subclasses, including allotypes, hinge variation, and glycosylation. The variation originating from allotypic polymorphisms in the immunoglobulin heavy gamma (IGHG) Fc domain is indicated with blue stars. Except for the star representing the variation in hinge length between IgG3 allotypes, each smaller blue star indicates amino acid variation at one particular residue in the constant domain. Glycans attached to N297 in Fc region are highly variable and the frequency of each glycan moiety on IgG antibodies in human serum is indicated [22] (reprinted with permission).*

**91**

**Figure 14.**

**Figure 13.**

*Analytical Characterization of Monoclonal Antibodies with Novel Fc Receptor-Based…*

individual peak size differences, each mAb is indicative of its unique separation profile (**Figure 13**). This study thus shows that TSKgel FcR-IIIA-NPR column can be used for the analysis of a variety of IgG subclasses for glycosylation profiling. The

Since the first FDA approvals of biosimilars in the USA in 2015, the interest continues to increase toward biosimilars. They are, in general, less costly to develop than the original or innovators. However, the biosimilar manufacturers are required to confirm the extent of similarity with the corresponding innovator. In the recent literature report [23], it was indeed confirmed that glycan microheterogeneity may play a critical role in effector function between the originals and biosimilars. In the study, it was shown that a biosimilar had a higher level of afucosylated glycans, resulting in a stronger FcγRIIIa binding affinity and increased ADCC activity. The study in the **Figure 14** also shows that the TSKgel FcR-IIIA-NPR column yielded dissimilar chromatographic pattern for biosimilars as compared to its innovator. Thus this column can be used for monitoring biosimilar consistent with the innovator biomolecule.

*Comparison of Roche's innovator Rituximab to its two biosimilar biotherapeutic forms. The figure also includes* 

*mogamulizumab (Poteligeo™) as an example of a completely afucosylated mAb.*

*Analysis of a variety of monoclonal antibodies on TSKgel FcR-IIIA-NPR.*

*DOI: http://dx.doi.org/10.5772/intechopen.95356*

results typically well correlate to ADCC activity.

#### *Analytical Characterization of Monoclonal Antibodies with Novel Fc Receptor-Based… DOI: http://dx.doi.org/10.5772/intechopen.95356*

individual peak size differences, each mAb is indicative of its unique separation profile (**Figure 13**). This study thus shows that TSKgel FcR-IIIA-NPR column can be used for the analysis of a variety of IgG subclasses for glycosylation profiling. The results typically well correlate to ADCC activity.

Since the first FDA approvals of biosimilars in the USA in 2015, the interest continues to increase toward biosimilars. They are, in general, less costly to develop than the original or innovators. However, the biosimilar manufacturers are required to confirm the extent of similarity with the corresponding innovator. In the recent literature report [23], it was indeed confirmed that glycan microheterogeneity may play a critical role in effector function between the originals and biosimilars. In the study, it was shown that a biosimilar had a higher level of afucosylated glycans, resulting in a stronger FcγRIIIa binding affinity and increased ADCC activity. The study in the **Figure 14** also shows that the TSKgel FcR-IIIA-NPR column yielded dissimilar chromatographic pattern for biosimilars as compared to its innovator. Thus this column can be used for monitoring biosimilar consistent with the innovator biomolecule.

#### **Figure 13.**

*Monoclonal Antibodies*

of different glycan molecules on the binding strength to Fc receptor can be arranged in the following order: Galactosylated (terminal) > Afucosylated > Sialylated (terminal) N-glycans in mAb. Similalry, retention time and ADCC activity is expected to be in the following order in the other forms of glycosylation patterns; A2G2 > A2G2S2 > A2G0, High mannose (HM) > FA2G2 and FA2G2S2 unless otherwise affected by any other factor. High mannose and A2G0 may be of similar activity. Complement-dependent cytotoxicity (CDC) is

**5. Glycosylation profiling of a variety of monoclonal antibodies**

are also gaining plenty of interest for use as biotherapeutics (**Figure 12**).

*A structural representation of the IgG subclasses and the variation within these subclasses, including allotypes, hinge variation, and glycosylation. The variation originating from allotypic polymorphisms in the immunoglobulin heavy gamma (IGHG) Fc domain is indicated with blue stars. Except for the star representing the variation in hinge length between IgG3 allotypes, each smaller blue star indicates amino acid variation at one particular residue in the constant domain. Glycans attached to N297 in Fc region are highly variable and the frequency of each glycan moiety on IgG antibodies in human serum is indicated [22] (reprinted with permission).*

Several commercially available IgGs were recently analyzed in-house using TSKgel FcR-IIIA-NPR column to compare their elution profiles. Almost all IgGs yielded the typical 3-peak separation profile although there was substantial difference in each peak height between IgGs. Generally, IgG1, IgG3 and IgG4 subclasses bind to TSKgel FcR-IIIA-NPR whereas IgG2 does not have affinity. Due to

IgG1 is the most abundant antibody in human body. Other antibodies are IgG2, IgG3, and IgG4 (**Figure 12**). Generally, IgG1, IgG3, and to some extent, IgG4 are formed against protein antigens. IgG2 is the major subclass formed against repetitive T cell-independent polysaccharide structures found on encapsulated bacteria [22]. Most of the biotherapeutics predominantly belong to IgG1 subclass. The antibodies from subclasses other than IgG1, as well as numerous engineered forms,

not significantly related to sugar structure [18].

**90**

**Figure 12.**

*Analysis of a variety of monoclonal antibodies on TSKgel FcR-IIIA-NPR.*

#### **Figure 14.**

*Comparison of Roche's innovator Rituximab to its two biosimilar biotherapeutic forms. The figure also includes mogamulizumab (Poteligeo™) as an example of a completely afucosylated mAb.*
