**10. Novelty of TSKgel FcR-IIIA-NPR column**

The mechanism of binding for IgG and other Fc engaging molecules is shown in the **Figure 28**. Complement component (C1q), Fc gamma receptors (FcγR), the Neonatal Fc receptor (FcRn), Tripartite motif 21 (Trim21), and Fc receptor-like (FcRL) molecules bind to various locations of mAb for the exertion of biological activity. For each ligand, the stoichiometric ratio of binding is also reported (Panel a). Recently, a biotinylated recombinant human FcRn immobilized to a Streptavidin Sepharose matrix and packed in a low pressure FPLC column has been introduced by Roche. A prepacked analytical protein-A affinity column (TSKgel ProteinA 5-PW) marketed by Tosoh also interacts with Fc region. Site of interaction of FcγR is separate from the site of interaction for FcRn or Protein A as seen in the **Figure 28** Panel b.

**101**

**Figure 29.**

**Figure 28.**

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

TSKgel FcR-IIIA-NPR and TSKgel Protein A-5PW columns were tested for binding affinity of mAb with and without N-glycan using surface plasmon resonance technique (**Figure 29**). Protein A affinity chromatography column showed similar binding to mAb regardless of N-glycan whereas TSKgel FcR-IIIA-NPR column did not bind to mAb without N-glycan, similarly to the result in the **Figure 20**. Thus, the FcR column is unique due to its capability to analyze mAbs solely on the basis of their glycosylation.

Here, we provide a quick preview of the preparative scale TSKgel FcR-IIIA-5PW column that will be commercially available soon. The preparative column is manufactured using the same recombinant FcγRIIIA protein ligand, however, the ligand in this column is bound to porous (~100 nm nominal pore size) polymethacrylate polymer base beads. The column is suited for mAb purification in a significantly larger scale (loading 0.5–5 mg) as compared to the analytical FcR column (loading ≤100 μg). Chromatographic profile in the **Figure 30**, panel A was obtained using U/HPLC instrument and the analytical FcR column whereas the panel B shows peak separation with the preparative FcR column connected to a FPLC instrument. The peak separation profiles are closely similar with both columns. However, the preparative scale column allows collection of much more material for further experimenta-

**11. Preparative scale purification of antibodies using FcR-based** 

*Selectivity of the modified recombinant Fc*γ*RIIIa ligand vs. Protein A ligand.*

tion such as glycan release, labeling and HILIC analysis, among other assays.

**chromatography technique**

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

*Interaction of IgG with Fc effector molecules and protein A.*

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

**Figure 28.**

*Monoclonal Antibodies*

**Figure 26.**

**Figure 27.**

flow-through. Fc fragment efficiently bound to the column and yielded three glycoform peaks similar to intact mAb (**Figure 27**). Same sample volumes from the control sample and digestion reaction mixture were loaded onto the column. Lower peak heights for the Fc fragment were due to loss of Fab (2 x 48 kDa) from the protein mass during analysis. Interestingly, slightly longer retention times were detected for Fc fragment peaks, thus suggesting more rigid conformational stability for the Fc fragment leading to stronger binding as compared to the intact mAb. In summary, this experiment confirms that fragment antibodies, as long as they contain intact unobstructed Fc region, can be tested using the FcR column.

*Schematic representation of monoclonal antibody fragmentation with papain.*

*Analysis of intact mAb and Fc fragment on TSKgel FcR-IIIA-NPR column.*

The mechanism of binding for IgG and other Fc engaging molecules is shown in the **Figure 28**. Complement component (C1q), Fc gamma receptors (FcγR), the Neonatal Fc receptor (FcRn), Tripartite motif 21 (Trim21), and Fc receptor-like (FcRL) molecules bind to various locations of mAb for the exertion of biological activity. For each ligand, the stoichiometric ratio of binding is also reported (Panel a). Recently, a biotinylated recombinant human FcRn immobilized to a Streptavidin Sepharose matrix and packed in a low pressure FPLC column has been introduced by Roche. A prepacked analytical protein-A affinity column (TSKgel ProteinA 5-PW) marketed by Tosoh also interacts with Fc region. Site of interaction of FcγR is separate from the site of interaction for FcRn or Protein A as seen in the **Figure 28**

**10. Novelty of TSKgel FcR-IIIA-NPR column**

**100**

Panel b.

*Interaction of IgG with Fc effector molecules and protein A.*

#### **Figure 29.**

*Selectivity of the modified recombinant Fc*γ*RIIIa ligand vs. Protein A ligand.*

TSKgel FcR-IIIA-NPR and TSKgel Protein A-5PW columns were tested for binding affinity of mAb with and without N-glycan using surface plasmon resonance technique (**Figure 29**). Protein A affinity chromatography column showed similar binding to mAb regardless of N-glycan whereas TSKgel FcR-IIIA-NPR column did not bind to mAb without N-glycan, similarly to the result in the **Figure 20**. Thus, the FcR column is unique due to its capability to analyze mAbs solely on the basis of their glycosylation.

### **11. Preparative scale purification of antibodies using FcR-based chromatography technique**

Here, we provide a quick preview of the preparative scale TSKgel FcR-IIIA-5PW column that will be commercially available soon. The preparative column is manufactured using the same recombinant FcγRIIIA protein ligand, however, the ligand in this column is bound to porous (~100 nm nominal pore size) polymethacrylate polymer base beads. The column is suited for mAb purification in a significantly larger scale (loading 0.5–5 mg) as compared to the analytical FcR column (loading ≤100 μg). Chromatographic profile in the **Figure 30**, panel A was obtained using U/HPLC instrument and the analytical FcR column whereas the panel B shows peak separation with the preparative FcR column connected to a FPLC instrument. The peak separation profiles are closely similar with both columns. However, the preparative scale column allows collection of much more material for further experimentation such as glycan release, labeling and HILIC analysis, among other assays.

**Figure 30.** *Separation of mAb glycoforms using (A) analytical FcR column and (B) preparative FcR column.*
