**4. Correlation between mAb retention time and ADCC activity**

In order to confirm correlation with ADCC activity and retention time, Rituximab as an example [20], was analyzed and three successive peaks from TSKgel FcR-IIIA-NPR column were collected. ADCC bioassay was performed for these peaks using the Promega ADCC reporter assay kit (**Figure 9**). In the assay, higher luminescence (RLU units), as compared to mAb concentration, denotes to stronger ADCC activity. As a measure of ADCC potency, EC50 values (mAb concentration with 50% of the maximal ADCC activity) were determined. RLU units for Rituximab sample prior to load and for three fractions were plotted as a function of their concentrations (μg/mL) (**Figure 9**, Panel B). The order of ADCC activity in the peaks is as follows: peak 3 > peak 2 > mAb > peak 1. The result thus clearly indicates that the peak 3 has the highest ADCC activity as compared to the other two peaks. This study proves that the increased retention

#### **Figure 9.**

*ADCC reporter bioassays for Rituximab and its glycoforms separated on TSKgel FcR-IIIA-NPR. Panel A: A typical 3-peak elution pattern for the mAb. Panel B: ADCC reporter bioassay for the mAb control and collected peaks from panel A. Panel C: EC50 values for the three peaks. (reprinted with permission).*

**89**

**Figure 11.**

**Figure 10.**

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

time for the peaks obtained from the U/HPLC assay indeed correlates with the

The core fucose can also exert a strong modulatory effect on the affinity for FcγRIIIa. **Figure 10** illustrates the location of a fucose molecule in the glycan moiety. In the mAb, the core fucose inhibits the carbohydrate contacts and decreases the binding affinity. Fucosylation of N-glycan thus reduces the affinity of mAb-FcR interaction by steric hindrance within the Fc cavity, obstructing the carbohydrate–

Afucosylated mAb binds effectively to FcγRIIIa ligand [19]. Subsequently, fucosylated and non-fucosylated mAbs were compared on the column where it was shown that deletion of core fucose significantly increased both retention time and

*Schematic representation of the core fucose location in the N-glycan moiety (reprinted with permission).*

*Effect of the core fucose on ADCC activity [20] (reprinted with permission).*

Sialic acid (N-acetylneuraminic acid) has a role in ADCC activity. Sialylation in the context of core fucosylation significantly decreased ADCC activity [21] as the sialic acid lowers the binding affinity to Fc receptor [19]. In absense of core fucosylation, sialylation doesn't have any significant impact on ADCC activity [21]. FcγRIIIa affinity chromatography yields longer retention by core fucosylation, terminal galactosylation and sialylation [18]. Overall, the effect

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

higher ADCC activity.

carbohydrate interaction [15].

the ADCC activity (**Figure 11**).

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

time for the peaks obtained from the U/HPLC assay indeed correlates with the higher ADCC activity.

The core fucose can also exert a strong modulatory effect on the affinity for FcγRIIIa. **Figure 10** illustrates the location of a fucose molecule in the glycan moiety. In the mAb, the core fucose inhibits the carbohydrate contacts and decreases the binding affinity. Fucosylation of N-glycan thus reduces the affinity of mAb-FcR interaction by steric hindrance within the Fc cavity, obstructing the carbohydrate– carbohydrate interaction [15].

Afucosylated mAb binds effectively to FcγRIIIa ligand [19]. Subsequently, fucosylated and non-fucosylated mAbs were compared on the column where it was shown that deletion of core fucose significantly increased both retention time and the ADCC activity (**Figure 11**).

Sialic acid (N-acetylneuraminic acid) has a role in ADCC activity. Sialylation in the context of core fucosylation significantly decreased ADCC activity [21] as the sialic acid lowers the binding affinity to Fc receptor [19]. In absense of core fucosylation, sialylation doesn't have any significant impact on ADCC activity [21]. FcγRIIIa affinity chromatography yields longer retention by core fucosylation, terminal galactosylation and sialylation [18]. Overall, the effect

**Figure 10.**

*Monoclonal Antibodies*

binding.

(HDX-MS) study using purified IgG glycovariants support this hypothesis. By the deuterium exchange mass spectrometry, it was noticed that the deuterium uptake increases in the peptide ranging from 245 P to 256 T in the following order: Peak 1 > Peak 2 > Peak 3. This result implicates that this particular peptide exhibits a more rigid conformation as the fraction of galactose units increase. Differential Scanning Calorimetry (DSC) experiment also proved that the peak 3 contains antibodies with the highest galactose content, and it exhibited the greatest denaturation enthalpy. This result thus suggests that the terminal galactose engages in non-covalent interaction with surrounding residues leading to increased conformational stability. The value of entropy change decreased as the content of galactose increased, suggesting a reduction of the conformational entropy of the antibody. More specifically, terminal galactose moiety seems to especially stabilize the mAb hinge region. In N-glycans containing galactose, the CH2 domain remains in more rigid conformation as compared to the agalactosylated (G0F) glycoform (i.e. no galactose). Overall, the number of terminal galactoses have the greatest impact on the binding affinity of mAb onto the column [19]. However, the other types of glycans such as fucose, mannose and sialic acid also affect the

**4. Correlation between mAb retention time and ADCC activity**

In order to confirm correlation with ADCC activity and retention time, Rituximab as an example [20], was analyzed and three successive peaks from TSKgel FcR-IIIA-NPR column were collected. ADCC bioassay was performed for these peaks using the Promega ADCC reporter assay kit (**Figure 9**). In the assay, higher luminescence (RLU units), as compared to mAb concentration, denotes to stronger ADCC activity. As a measure of ADCC potency, EC50 values (mAb concentration with 50% of the maximal ADCC activity) were determined. RLU units for Rituximab sample prior to load and for three fractions were plotted as a function of their concentrations (μg/mL) (**Figure 9**, Panel B). The order of ADCC activity in the peaks is as follows: peak 3 > peak 2 > mAb > peak 1. The result thus clearly indicates that the peak 3 has the highest ADCC activity as compared to the other two peaks. This study proves that the increased retention

*ADCC reporter bioassays for Rituximab and its glycoforms separated on TSKgel FcR-IIIA-NPR. Panel A: A typical 3-peak elution pattern for the mAb. Panel B: ADCC reporter bioassay for the mAb control and collected* 

*peaks from panel A. Panel C: EC50 values for the three peaks. (reprinted with permission).*

**88**

**Figure 9.**

*Schematic representation of the core fucose location in the N-glycan moiety (reprinted with permission).*

**Figure 11.**

*Effect of the core fucose on ADCC activity [20] (reprinted with permission).*

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 not significantly related to sugar structure [18].
