**2.3 Fc**γ**R subclasses**

FcγRs are divided into three subclasses, abbreviated as FcγRI, FcγRII and FcγRIII. Extracellular regions of all the FcγRs are extremely homologous, whereas the

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

cytoplasmic domains differ considerably from each other [12]. FcγRI exhibits the high-

*Schematic overview of human IgG1 with a detailed sequence view into the hinge region [10] (reprinted with* 

ing. FcγRIII (also known as CD16) is a cluster of differentiation molecule found on the

FcγRIII exists in two different isoforms, (a) FcγRIIIa or CD16a and (b) FcγRIIIb or CD16b. Both forms take part in intracellular signal transduction. Two nearly identical genes in human encode these two isoforms. FcγRIIIa is a 50–65 kDa type-1 transmembrane protein whereas FcγRIIIb is a 48 kDa glycosylphosphatidylinositol (GPI)-anchored protein. This chapter focuses on the modified recombinant FcγRIIIa protein ligand, immobilized on a polymethacrylate stationary phase and packed into an analytical chromatography column that can be used for characteriza-

Post-translational modifications, particularly glycosylation, of both IgG antibodies and Fcγ receptors modulate the affinity of their interaction. N-glycan (**Figure 2**) is a well-defined complex biantennary structure composed of a core hepta-saccharide, made up of N-acetylglucosamine (GlcNAc) and mannose, followed by variable additions of galactose, sialic acid (N-acetylneuraminic acid),

The attached glycans play various crucial roles on the function of immunoglobulins. Fc sialylation prolongs serum half-life of therapeutic antibodies [14]. **Figure 3** shows details of the structure of the glycosylated Fc fragment complexed to a FcγRIIIa receptor [15]. In non-fucosylated mAb, the carbohydrate-carbohydrate interactions increase binding affinity between N-Glycan of IgG-Fc and N-Glycan of FcR (KD = 7.2 × 10−9 M) while in fucosylated mAb the carbohydrate-carbohydrate interaction is weekened or non-existent depending on the extent of steric hindrance

surface of natural killer (NK) cells, neutrophils, monocytes and macrophages.

M−1 whereas FcγRII and FcγRIII show a weaker affinity

M−1. Receptor clustering is essential for FcγR signal-

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

est affinity for IgG, Ka 108

**Figure 1.**

*permission).*

**2.4 Fc**γ**RIII isoforms**

[13] for monomeric IgG, Ka ≤ 107

–109

tion of antibodies based on their N-glycan content on Asn297.

**2.5 Fc**γ**RIIIa and glycosylation mode of IgG**

fucose, and bisecting GlcNAc residues [11].

of the fucose moiety (KD = 3.0 × 10−7 M) [15].

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

#### **Figure 1.**

*Monoclonal Antibodies*

**2.1 Fc**γ**Rs**

**2. Brief overview of Fc receptor**

receptors do not bind to IgA or IgM [9].

**2.2 Fc**γ **receptor binding to IgG**

chromatography ligands are available as resins and pre-packed columns for purification at various scales. Similarly, different types of U/HPLC affinity chromatography columns are available for analytical characterization and quality control. This chapter focuses on a recently introduced novel FcR-based affinity column, TSKgel-FcR-IIIA-NPR, that enables chromatographic characterization of mAbs based on their N-glycan content attached to a highly conserved Asn-297 in Fc region.

A brief overview of the Fc Receptor (FcR) structure and function is provided to best understand the chromatography principle of the column discussed in this chapter. FcR proteins belong to immunoglobulin (Ig) superfamily [1]. Interest in FcRs for biotherapeutic research has gained momentum since 1980s. The purification of FcR from the glycoprotein fraction of the placental membranes by chromatography was reported in 1982 [2]. A functional 40 kDa FcR, with low affinity for native IgG, was purified from the human peripheral nerve extract using F(ab)2 fragments of mAb against placental FcR as affinity agent in 1989 [3]. Important role of FcR in IgG distribution to the brain [4], inhibition of cell activation [5] and in enhancement and suppression of the effector function [6] have also been reported. Overall, it became

The FcRs binding to immunoglobulin G (IgG) are known as Fc-gamma receptors (FcγR). FcγRs play essential role in immunity, inflammatory and infectious diseases [7]. Immune enhancement and suppression are influenced by binding to these FcγRs [6]. Additional interaction between hyaluronic acid (HA) and sialic acids on immune cells helps to optimize the FcR-mediated effector function [8]. Fcγ

Typical IgG is Y-shaped protein of ~150 kDa in size, containing two heavy chains

and two light chains (**Figure 1**). The heavy chain (HC) contains three constant domains (CH1–CH3) and a variable domain (VH) with three complementaritydetermining regions (CDRs). The light chain (LC) has only one constant domain (CL) and a variable domain (VL) with CDRs. The Fd consists of VH and CH1. LC and Fd together form the antigen binding fragment (Fab). The CH2 domain of each heavy chain of IgG has a highly conservative asparagine (N) residue at position 297 (Asn297 or N297) that is almost invariably glycosylated. Fcγ receptor binding site is located near the hinge region of IgG, close to N297 in the CH2 domain. The most flexible portion of the hinge region is between CH1 and CH2 domains of a heavy chain. The four chains are covalently connected via disulfide bridges [10]. Fraction crystallizable (Fc) is composed of CH2 and CH3 domains of the two heavy chains. The highly conserved glycan moiety at position N297 infers structural changes to the Fc-region required for binding to FcγR. Subtle differences in the glycan composition at this site, thus, can affect the conformational rigidity of the Fc-structure,

and may also alter the interaction with FcγR by direct contact [11].

FcγRs are divided into three subclasses, abbreviated as FcγRI, FcγRII and FcγRIII.

Extracellular regions of all the FcγRs are extremely homologous, whereas the

evident that FcRs are important for numerous biological functions.

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**2.3 Fc**γ**R subclasses**

*Schematic overview of human IgG1 with a detailed sequence view into the hinge region [10] (reprinted with permission).*

cytoplasmic domains differ considerably from each other [12]. FcγRI exhibits the highest affinity for IgG, Ka 108 –109 M−1 whereas FcγRII and FcγRIII show a weaker affinity [13] for monomeric IgG, Ka ≤ 107 M−1. Receptor clustering is essential for FcγR signaling. FcγRIII (also known as CD16) is a cluster of differentiation molecule found on the surface of natural killer (NK) cells, neutrophils, monocytes and macrophages.

### **2.4 Fc**γ**RIII isoforms**

FcγRIII exists in two different isoforms, (a) FcγRIIIa or CD16a and (b) FcγRIIIb or CD16b. Both forms take part in intracellular signal transduction. Two nearly identical genes in human encode these two isoforms. FcγRIIIa is a 50–65 kDa type-1 transmembrane protein whereas FcγRIIIb is a 48 kDa glycosylphosphatidylinositol (GPI)-anchored protein. This chapter focuses on the modified recombinant FcγRIIIa protein ligand, immobilized on a polymethacrylate stationary phase and packed into an analytical chromatography column that can be used for characterization of antibodies based on their N-glycan content on Asn297.

#### **2.5 Fc**γ**RIIIa and glycosylation mode of IgG**

Post-translational modifications, particularly glycosylation, of both IgG antibodies and Fcγ receptors modulate the affinity of their interaction. N-glycan (**Figure 2**) is a well-defined complex biantennary structure composed of a core hepta-saccharide, made up of N-acetylglucosamine (GlcNAc) and mannose, followed by variable additions of galactose, sialic acid (N-acetylneuraminic acid), fucose, and bisecting GlcNAc residues [11].

The attached glycans play various crucial roles on the function of immunoglobulins. Fc sialylation prolongs serum half-life of therapeutic antibodies [14]. **Figure 3** shows details of the structure of the glycosylated Fc fragment complexed to a FcγRIIIa receptor [15]. In non-fucosylated mAb, the carbohydrate-carbohydrate interactions increase binding affinity between N-Glycan of IgG-Fc and N-Glycan of FcR (KD = 7.2 × 10−9 M) while in fucosylated mAb the carbohydrate-carbohydrate interaction is weekened or non-existent depending on the extent of steric hindrance of the fucose moiety (KD = 3.0 × 10−7 M) [15].

#### **Figure 2.**

*A schematic representation of a common N-glycan structure where blue squares denote to GlcNAc, green circles denote to mannose, yellow circles denote to galactose, purple diamonds denote to sialic acid (N-acetylneuraminic acid) and red triangle denote to fucose (reprinted with permission).*

#### **Figure 3.**

*Crystal structure of glycosylated Fc-Fc*γ*RIIIa complex. (A) Top and side views of the structure of the glycosylated Fc-Fc*γ*RIIIa complex. The Fc chains are shown in blue and magenta and the receptor in cyan. The oligosaccharides are depicted as ball-and-stick representation. (B) View on the interaction interface between afucosylated Fc fragment and glycosylated Fc receptor. Chain A of the Fc fragment is shown in blue, the Fc receptor in cyan. Hydrogen bonds are presented as dashed lines with distance between donor and acceptor atoms. (C) View on the interaction interface between fucosylated Fc fragment and glycosylated Fc receptor. Chain A of the Fc fragment is shown in magenta, the Fc receptor in dark violet. Core fucose of fucosylated Fc is highlighted in yellow [15]. (Reprinted with permission).*

The glycosylation of Fc part is prerequisite for its affinity to FcR. Therapeutic monoclonal antibodies recognize specific cell surface-expressed antigens in malfunctioning cells (e.g. cancer cells) and elicit immune effector functions such as Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) (**Figure 4**). Glycan composition at conserved N297 in IgG largely affects the binding affinity, thus regulating ADCC activity.

FcγRIIIa protein contacts Fc portion of IgG (to both CH2 regions) and to attached glycans. The glycans from FcγRIIIa side are also apparently in contact with glycans from IgG-Fc. The binding is asymmetrical in nature as revealed from the co-crystal structure of Fc-FcγRIII complex [16], although the stoichiometry of binding is 1:1. Lack of fucosylation in core Fc-glycan dramatically increases the ADCC activity due to enhanced binding affinity of FcγRIIIa to IgG [17].

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

TSKgel FcR-IIIA-NPR affinity column contains non-porous polymethacrylate base beads as stationary phase. The ligand is a modified recombinant non-glycosylated FcγRIIIa of 20 kDa, produced in *E. coli* expression system. Recombinant FcγRIIIa ligand has eight amino acid substitutions as compared to its wild-type form. These changes were necessary for stabilization of the ligand structure [18]. The 2.7 Å crystal structure of recombinant FcγRIIIa protein verifies the molecular basis of the IgG-FcR complex formation. No significant difference was found between the crystal structures of glycosylated wild-type FcγRIIIa expressed in human embryonic kidney (HEK) cells vs. non-glycosylated recombinant FcγRIIIa from *E. coli* (**Figure 5**). This confirms suitability of *E. coli*-produced non-glycosylated FcγRIIIa to be used as an affinity ligand in a chromatography resin. Notably, no direct contact of the terminal Asn297 N-glycan galactose of IgG and modified non-glycosylated FcγRIIIa was

observed [19]. Both proteins were crystallized as complex with Fc [19].

*Schematic representation of the antibody-Fc receptor interaction resulting in ADCC activity.*

The dimension of the TSKgel FcR-IIIA-NPR column is 4.6 mm ID × 7.5 cm (l) with a total bed volume of 1.25 mL. Polymethacrylate-based matrix is composed of non-porous material with ~5 μm particle size. Maximum pressure limit of the column is 90 bar (9 MPa). The column is suitable for both HPLC and UHPLC instrument settings. The recommended run temperature is 15–25°C. The operational pH range is from pH 4 to 8. In general, most monoclonal antibodies bind effectively on the column at pH 6.5. Typically, 50 mM ammonium citrate (or ammonium acetate) buffer is used. A linear pH gradient from pH 6.5 to 4.5 over 16 column volumes (CV) at the flow rate of 1.0 mL/min is recommended. **Figure 6** shows a typical threepeak chromatographic profile for a monoclonal antibody in these settings. Sodium chloride can be added to buffer to enhance the separation if needed. Longer retention time indicates stronger mAb affinity to the ligand. However, as to the general composition of glycans, it should be noted that all the three peaks still contain a mixture of glycoforms with variable amounts of galactose and other sugar molecules (**Figure 7**). From a related experiment (**Figure 7**), the three peaks were collected for glycan analysis and ADCC activity assay. Determination of the glycan structures revealed that the retention time increase correlates with increased number of the terminal galactose. Terminal galactose tends to stabilize conformation of the Fc region, providing tighter binding onto FcγRIIIa affinity ligand [20]. However, FcR column is not designed for quantitation of only galactose but to obtain a more general understanding of the variation in distribution of the glycan content among the

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

**3. TSKgel FcR-IIIA-NPR affinity column**

**Figure 4.**

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

**Figure 4.**

*Monoclonal Antibodies*

**Figure 2.**

**Figure 3.**

**84**

regulating ADCC activity.

*highlighted in yellow [15]. (Reprinted with permission).*

The glycosylation of Fc part is prerequisite for its affinity to FcR. Therapeutic

monoclonal antibodies recognize specific cell surface-expressed antigens in malfunctioning cells (e.g. cancer cells) and elicit immune effector functions such as Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) (**Figure 4**). Glycan composition at conserved N297 in IgG largely affects the binding affinity, thus

*Crystal structure of glycosylated Fc-Fc*γ*RIIIa complex. (A) Top and side views of the structure of the glycosylated Fc-Fc*γ*RIIIa complex. The Fc chains are shown in blue and magenta and the receptor in cyan. The oligosaccharides are depicted as ball-and-stick representation. (B) View on the interaction interface between afucosylated Fc fragment and glycosylated Fc receptor. Chain A of the Fc fragment is shown in blue, the Fc receptor in cyan. Hydrogen bonds are presented as dashed lines with distance between donor and acceptor atoms. (C) View on the interaction interface between fucosylated Fc fragment and glycosylated Fc receptor. Chain A of the Fc fragment is shown in magenta, the Fc receptor in dark violet. Core fucose of fucosylated Fc is* 

*A schematic representation of a common N-glycan structure where blue squares denote to GlcNAc, green circles denote to mannose, yellow circles denote to galactose, purple diamonds denote to sialic acid* 

*(N-acetylneuraminic acid) and red triangle denote to fucose (reprinted with permission).*

FcγRIIIa protein contacts Fc portion of IgG (to both CH2 regions) and to attached glycans. The glycans from FcγRIIIa side are also apparently in contact with glycans from IgG-Fc. The binding is asymmetrical in nature as revealed from the co-crystal structure of Fc-FcγRIII complex [16], although the stoichiometry of binding is 1:1. Lack of fucosylation in core Fc-glycan dramatically increases the

ADCC activity due to enhanced binding affinity of FcγRIIIa to IgG [17].

*Schematic representation of the antibody-Fc receptor interaction resulting in ADCC activity.*

#### **3. TSKgel FcR-IIIA-NPR affinity column**

TSKgel FcR-IIIA-NPR affinity column contains non-porous polymethacrylate base beads as stationary phase. The ligand is a modified recombinant non-glycosylated FcγRIIIa of 20 kDa, produced in *E. coli* expression system. Recombinant FcγRIIIa ligand has eight amino acid substitutions as compared to its wild-type form. These changes were necessary for stabilization of the ligand structure [18]. The 2.7 Å crystal structure of recombinant FcγRIIIa protein verifies the molecular basis of the IgG-FcR complex formation. No significant difference was found between the crystal structures of glycosylated wild-type FcγRIIIa expressed in human embryonic kidney (HEK) cells vs. non-glycosylated recombinant FcγRIIIa from *E. coli* (**Figure 5**). This confirms suitability of *E. coli*-produced non-glycosylated FcγRIIIa to be used as an affinity ligand in a chromatography resin. Notably, no direct contact of the terminal Asn297 N-glycan galactose of IgG and modified non-glycosylated FcγRIIIa was observed [19]. Both proteins were crystallized as complex with Fc [19].

The dimension of the TSKgel FcR-IIIA-NPR column is 4.6 mm ID × 7.5 cm (l) with a total bed volume of 1.25 mL. Polymethacrylate-based matrix is composed of non-porous material with ~5 μm particle size. Maximum pressure limit of the column is 90 bar (9 MPa). The column is suitable for both HPLC and UHPLC instrument settings. The recommended run temperature is 15–25°C. The operational pH range is from pH 4 to 8. In general, most monoclonal antibodies bind effectively on the column at pH 6.5. Typically, 50 mM ammonium citrate (or ammonium acetate) buffer is used. A linear pH gradient from pH 6.5 to 4.5 over 16 column volumes (CV) at the flow rate of 1.0 mL/min is recommended. **Figure 6** shows a typical threepeak chromatographic profile for a monoclonal antibody in these settings. Sodium chloride can be added to buffer to enhance the separation if needed. Longer retention time indicates stronger mAb affinity to the ligand. However, as to the general composition of glycans, it should be noted that all the three peaks still contain a mixture of glycoforms with variable amounts of galactose and other sugar molecules (**Figure 7**).

From a related experiment (**Figure 7**), the three peaks were collected for glycan analysis and ADCC activity assay. Determination of the glycan structures revealed that the retention time increase correlates with increased number of the terminal galactose. Terminal galactose tends to stabilize conformation of the Fc region, providing tighter binding onto FcγRIIIa affinity ligand [20]. However, FcR column is not designed for quantitation of only galactose but to obtain a more general understanding of the variation in distribution of the glycan content among the

**Figure 5.**

*Crystal structures of a recombinant non-glycosylated FcR ligand (Panel a) and a glycosylated native form (Panel b) (reprinted with permission).*

**Figure 6.**

*A typical 3-peak elution profile for a monoclonal antibody using a TSKgel FcR-IIIA-NPR column with correlation to ADCC activity.*

three peaks. Although added galactose increases retention time, other factors (as explained below) also affect the mAb binding affinity.

The results shown in the **Figure 7** support the binding model presented for galactose in the **Figure 8**. The crystal structure, basis of a cartoon model in the **Figure 8**, surprisingly did not show direct contact of the galactose units with the receptor that could more easily explain galactose effect on the affinity for FcγRIIIa. Instead, based on the evidence reported in literature so far, it has been proposed that the galactose moiety can influence the dynamic and conformational assembly of IgG-Fc. Hydrogen-deuterium exchange mass spectrometry

**87**

**Figure 8.**

*(reprinted with permission).*

**Figure 7.**

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

*Correlation between the number of galactose units and retention time [20] (reprinted with permission).*

*Conformational entropy modulated by galactose content controls the binding affinity of IgG to Fc*γ*RIIIa [19].* 

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

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

**Figure 7.** *Correlation between the number of galactose units and retention time [20] (reprinted with permission).*

**Figure 8.**

*Conformational entropy modulated by galactose content controls the binding affinity of IgG to Fc*γ*RIIIa [19]. (reprinted with permission).*

*Monoclonal Antibodies*

**86**

**Figure 6.**

**Figure 5.**

*(Panel b) (reprinted with permission).*

*correlation to ADCC activity.*

three peaks. Although added galactose increases retention time, other factors (as

*A typical 3-peak elution profile for a monoclonal antibody using a TSKgel FcR-IIIA-NPR column with* 

*Crystal structures of a recombinant non-glycosylated FcR ligand (Panel a) and a glycosylated native form* 

The results shown in the **Figure 7** support the binding model presented for galactose in the **Figure 8**. The crystal structure, basis of a cartoon model in the **Figure 8**, surprisingly did not show direct contact of the galactose units with the receptor that could more easily explain galactose effect on the affinity for FcγRIIIa. Instead, based on the evidence reported in literature so far, it has been proposed that the galactose moiety can influence the dynamic and conformational assembly of IgG-Fc. Hydrogen-deuterium exchange mass spectrometry

explained below) also affect the mAb binding affinity.

(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 binding.
