1. Introduction

Antibodies belong to a family of globular proteins called immunoglobulins [1]. Immunoglobulin G (IgG) is the most common. Eighty percent of all the antibodies present in the blood are IgG [2]. IgG is a relatively large molecule (approx. 150 kDa). It has four subclasses, which are IgG1, IgG2, IgG3, and IgG4 [3]. Monoclonal antibodies are antibodies derived from one unique B cell clone [4]. They have single antigenic determinant specificities [5]. Monoclonal antibodies are screened and isolated by special procedures, expressed, and purified [6]. Monoclonal antibodies, particularly IgG1, have tremendous application in biotherapeutics. Other subclasses such as IgG2 and IgG4 are also used as biotherapeutic, and interest in these two antibody classes is also increasing. As for today, IgG1 comprise most of the mAb biotherapeutic drugs in the market. Twenty-nine new mAbs are presently undergoing late-stage clinical trials, including human and humanized IgG1, IgG2, and IgG4 molecules [7]. Few IgG2 and IgG4 drugs are already available such as OKT3 (Muronomab-CD3), a murine IgG2a drug from Johnson & Johnson (1986), Bexxar (Tositumomab-I-131), and a murine IgG2a drug radiolabeled with I-131 from Corixa/GSK (2003). IgG4 antibodies are evolving as an important class of cancer immunotherapies [8].

analysis of the mAb. Detection wavelength of 280 nm is commonly used. SEC combined with multi-angle light scattering is one method for the characterization of the molar mass distribu-

Separation of Monoclonal Antibodies by Analytical Size Exclusion Chromatography

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In this chapter, certain aspects of size exclusion chromatography and its use in the analytical purification or separation of monoclonal antibodies are discussed. Secondary interactions, effects of particle size and pore size on the SEC separation, particularly in resolving monomer peak from dimer and fragment peaks with a better resolution are also discussed. Selection of the right SEC column is critical to achieve the goal of separation. The calibration curve has a very important role in this regard. Since there are different kinds of proteins differing in structure and shape, many vendors provide calibration curves using globular protein standards, branched standards, and linear standards, so that the separation range can ideally be

Reproducible separation of a monomer from its dimer and other high molecular weight (HMW) impurities, fragments, and other low molecular weight (LMW) impurities is needed during the purification of the mAb biotherapeutic. Optimized particle chemistry and packing of SEC column help in this regard. mAb analysis using a mobile phase containing an appropriate amount of selected organic solvents such as isopropyl alcohol (IPA) may be needed to prevent alteration of peak retention time, poor peak shape, or resolution. Few examples are shown to elaborate this. Digestion with papain or pepsin is commonly applied to obtain antibody fragments without the loss of activity—this is discussed in the context of selecting right SEC columns from a variety of particle sizes, pore sizes, and dimensions. Forced degradation studies are needed to assess the stability of the protein, to understand the mechanism of degradation by oxidation, heat, light, or hydrolysis. Forced degradation study by SEC is separately discussed in Section 2.7. Interest in the accurate molecular weight analysis of intact monoclonal antibody IgG1 by SEC using MSfriendly mobile phases is increasing, and it is discussed in Section 4. Section 5 focuses mostly on the use of chromatographic methods which are orthogonal or complimentary size exclusion and useful to detect the protein heterogeneity. The use of ultra HPLC is needed for fast separation, and many methods already developed in HPLC need to be easily transferred to UHPLC. Section 6 briefly discusses the usefulness of a SEC column compatible to both HPLC and UHPLC instruments by easy method transfer. Section 7 is about desalting of mAb or any biopolymer solution, not by dialysis membrane or spin column but by using an analytical SEC HPLC column. The use of SEC column in hydrophilic liquid chromatography (HILIC) mode is interesting and can separate the nucleobases (Section 8). This chapter ends with few remarks about the

tion of mAb, ADC, and other biomolecules [12].

interpreted under the chromatographic conditions.

tips and tricks for size exclusion chromatography.

2.1. Size exclusion chromatography

antibodies

2. Size exclusion chromatography and purification of monoclonal

Size exclusion chromatography uses a molecular sieving retention mechanism [13], based on differences in the hydrodynamic radii or differences in size of analytes such as proteins. Large

Size exclusion chromatography (SEC) is a powerful analytical tool for the separation of monoclonal antibodies and other proteins [9]. SEC, as a strategy for the isolation and purification of antibodies, is not new; in 1989, high-resolution Superose 6 HR 10/30 fast protein liquid chromatography (FPLC) columns were used. [10]. Since then, many researchers used SEC for the purification of antibodies. The literature search for the number of "publications on the purification of monoclonal antibodies by size exclusion chromatography" [11] shows that between 1983 and 2003, there was a surge of research in this regard (Figure 1).

For the large-scale purification of monoclonal antibody biotherapeutics, Protein A is commonly used as the primary capture step. Following the use of Protein A chromatography, SEC is used to characterize the Protein A purified fractions. Size exclusion chromatography (SEC) is primarily used for the separation in analytical HPLC and for routine quality control

Figure 1. Number of publications dealing with size exclusion chromatography over the years.

analysis of the mAb. Detection wavelength of 280 nm is commonly used. SEC combined with multi-angle light scattering is one method for the characterization of the molar mass distribution of mAb, ADC, and other biomolecules [12].

In this chapter, certain aspects of size exclusion chromatography and its use in the analytical purification or separation of monoclonal antibodies are discussed. Secondary interactions, effects of particle size and pore size on the SEC separation, particularly in resolving monomer peak from dimer and fragment peaks with a better resolution are also discussed. Selection of the right SEC column is critical to achieve the goal of separation. The calibration curve has a very important role in this regard. Since there are different kinds of proteins differing in structure and shape, many vendors provide calibration curves using globular protein standards, branched standards, and linear standards, so that the separation range can ideally be interpreted under the chromatographic conditions.

Reproducible separation of a monomer from its dimer and other high molecular weight (HMW) impurities, fragments, and other low molecular weight (LMW) impurities is needed during the purification of the mAb biotherapeutic. Optimized particle chemistry and packing of SEC column help in this regard. mAb analysis using a mobile phase containing an appropriate amount of selected organic solvents such as isopropyl alcohol (IPA) may be needed to prevent alteration of peak retention time, poor peak shape, or resolution. Few examples are shown to elaborate this. Digestion with papain or pepsin is commonly applied to obtain antibody fragments without the loss of activity—this is discussed in the context of selecting right SEC columns from a variety of particle sizes, pore sizes, and dimensions. Forced degradation studies are needed to assess the stability of the protein, to understand the mechanism of degradation by oxidation, heat, light, or hydrolysis. Forced degradation study by SEC is separately discussed in Section 2.7. Interest in the accurate molecular weight analysis of intact monoclonal antibody IgG1 by SEC using MSfriendly mobile phases is increasing, and it is discussed in Section 4. Section 5 focuses mostly on the use of chromatographic methods which are orthogonal or complimentary size exclusion and useful to detect the protein heterogeneity. The use of ultra HPLC is needed for fast separation, and many methods already developed in HPLC need to be easily transferred to UHPLC. Section 6 briefly discusses the usefulness of a SEC column compatible to both HPLC and UHPLC instruments by easy method transfer. Section 7 is about desalting of mAb or any biopolymer solution, not by dialysis membrane or spin column but by using an analytical SEC HPLC column. The use of SEC column in hydrophilic liquid chromatography (HILIC) mode is interesting and can separate the nucleobases (Section 8). This chapter ends with few remarks about the tips and tricks for size exclusion chromatography.
