2. Size exclusion chromatography and purification of monoclonal antibodies

#### 2.1. Size exclusion chromatography

1. Introduction

134 Antibody Engineering

class of cancer immunotherapies [8].

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

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

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

1983 and 2003, there was a surge of research in this regard (Figure 1).

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

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 sample molecules cannot penetrate or only partially penetrate the pores of the stationary phase. So, the larger molecules elute first and smaller molecules elute later, the order of elution being a function of the size.

a stronger solvent. This is because SEC is purely a separation technique based on the permeability of the protein through the pores. Any factor which will prevent the free passage through the pores will result into a nonideal SEC condition. These interactions between proteins and the stationary phases are called secondary interactions. The secondary interactions are based on charge-related interactions or hydrophobicity-related interactions. The secondary interaction coming from the free silanol groups present on the silica surface of the stationary phase is often protected by a diol-bonded coating on the stationary phase which shields the

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In a real-world situation, any secondary interaction between the stationary phase and the proteins including monoclonal antibodies needs to be taken care of by selecting the right column where the stationary phase is effectively coated, and free silanol groups are inaccessible to proteins. Further method development by optimizing the chromatographic conditions may be needed to get the best separation. Ionic and hydrophobic interactions between the sample and the column packing material can be avoided by controlling the ionic strength. A general rule of thumb is that low ionic strength (<0.1 M) may induce chargerelated secondary interactions and high ionic strength (>1.0 M) may lead to hydrophobicityrelated secondary interactions, while the concept of low or high concentration may vary from mAb to mAb depending on the nature of the individual one. For each protein sample, there will be an optimal buffer type and salt concentration for the best separation that results in the highest resolution and recovery. This can be found out by trial and error approach only. If a sticky protein comes into contact with the stationary phase (dotted line in the figure below), it may undergo a conformational change; the binding constant of the conformationally changed protein can be so strong that it would not elute out of the column (Figure 3). The use of additives may be needed. Arginine prevents binding to the surface [15]. Similarly, a number of other additives may also be used to minimize the secondary interactions. The use of isopropyl alcohol (IPA) as another additive to avoid nonspecific interaction and the

reproducibility of the analysis is somewhat discussed in the Section 2.5.

out by trial-and-error type of experiments with a variety of additives.

Figure 3. Binding of a protein to the stationary phase and the influence of arginine.

There is no universal protocol for working with additives to avoid nonspecific interactions for all proteins. Chromatographic conditions preventing secondary interactions need to be found

silica surface from such action [14].

SEC is the only mode of chromatography where theoretically there is no interaction of the analyte with a stationary phase. The whole process of partitioning or separating the different molecular species is due to the entropy factor and not due to adsorption, ΔH being equal to zero. Pure Silica particles are most commonly used as base material in this type of chromatography of biomolecules. Since the separation of the biomolecule by SEC will depend on its hydrodynamic radii, two proteins of the same molecular weight (such as 70 kDa) may elute at two different retention times, if there is a difference in their hydrodynamic radii (Figure 2). So, any factor at any stage of purification, affecting the shape of the protein, will affect the elution volume or retention time.

Unless otherwise mentioned, SEC analyses discussed in this chapter were carried out using the mobile phase 100 mM KH2PO4/Na2HPO4, pH 6.7, 100 mM Na2SO4, 0.05% NaN3. Agilent 1100, Agilent 1200 HPLC and Thermo Ultimate 3000 UHPLC systems and associated software were used for integration and peak analysis. Sodium azide (NaN3) was used as an antibacterial agent to prevent fouling of the phosphate buffer. Detection wavelength was 280 nm unless otherwise mentioned. Flow rates and injection volume of the sample are varied as needed. Reproducibility for calculation of % relative standard deviation (RSD) was based on 10 consecutive injections. Linearity of both monomer and dimer during loading study was calculated based on peak areas versus total material loaded during injection. Please refer to individual chromatograms for the respective chromatographic conditions.

#### 2.2. SEC and secondary interaction

As mentioned earlier, among all modes of chromatography, it is only during the size exclusion chromatography where the analyst does not demonstrate any kind of interaction with the stationary phase. During all other chromatographic modes, an analyst demonstrates some kind of interaction between the protein and the stationary phase, followed by the elution using

Figure 2. Comparison of globular and rod-shaped proteins.

a stronger solvent. This is because SEC is purely a separation technique based on the permeability of the protein through the pores. Any factor which will prevent the free passage through the pores will result into a nonideal SEC condition. These interactions between proteins and the stationary phases are called secondary interactions. The secondary interactions are based on charge-related interactions or hydrophobicity-related interactions. The secondary interaction coming from the free silanol groups present on the silica surface of the stationary phase is often protected by a diol-bonded coating on the stationary phase which shields the silica surface from such action [14].

sample molecules cannot penetrate or only partially penetrate the pores of the stationary phase. So, the larger molecules elute first and smaller molecules elute later, the order of elution

SEC is the only mode of chromatography where theoretically there is no interaction of the analyte with a stationary phase. The whole process of partitioning or separating the different molecular species is due to the entropy factor and not due to adsorption, ΔH being equal to zero. Pure Silica particles are most commonly used as base material in this type of chromatography of biomolecules. Since the separation of the biomolecule by SEC will depend on its hydrodynamic radii, two proteins of the same molecular weight (such as 70 kDa) may elute at two different retention times, if there is a difference in their hydrodynamic radii (Figure 2). So, any factor at any stage of purification, affecting the shape of the protein, will affect the

Unless otherwise mentioned, SEC analyses discussed in this chapter were carried out using the mobile phase 100 mM KH2PO4/Na2HPO4, pH 6.7, 100 mM Na2SO4, 0.05% NaN3. Agilent 1100, Agilent 1200 HPLC and Thermo Ultimate 3000 UHPLC systems and associated software were used for integration and peak analysis. Sodium azide (NaN3) was used as an antibacterial agent to prevent fouling of the phosphate buffer. Detection wavelength was 280 nm unless otherwise mentioned. Flow rates and injection volume of the sample are varied as needed. Reproducibility for calculation of % relative standard deviation (RSD) was based on 10 consecutive injections. Linearity of both monomer and dimer during loading study was calculated based on peak areas versus total material loaded during injection. Please refer to individual

As mentioned earlier, among all modes of chromatography, it is only during the size exclusion chromatography where the analyst does not demonstrate any kind of interaction with the stationary phase. During all other chromatographic modes, an analyst demonstrates some kind of interaction between the protein and the stationary phase, followed by the elution using

chromatograms for the respective chromatographic conditions.

being a function of the size.

136 Antibody Engineering

elution volume or retention time.

2.2. SEC and secondary interaction

Figure 2. Comparison of globular and rod-shaped proteins.

In a real-world situation, any secondary interaction between the stationary phase and the proteins including monoclonal antibodies needs to be taken care of by selecting the right column where the stationary phase is effectively coated, and free silanol groups are inaccessible to proteins. Further method development by optimizing the chromatographic conditions may be needed to get the best separation. Ionic and hydrophobic interactions between the sample and the column packing material can be avoided by controlling the ionic strength. A general rule of thumb is that low ionic strength (<0.1 M) may induce chargerelated secondary interactions and high ionic strength (>1.0 M) may lead to hydrophobicityrelated secondary interactions, while the concept of low or high concentration may vary from mAb to mAb depending on the nature of the individual one. For each protein sample, there will be an optimal buffer type and salt concentration for the best separation that results in the highest resolution and recovery. This can be found out by trial and error approach only. If a sticky protein comes into contact with the stationary phase (dotted line in the figure below), it may undergo a conformational change; the binding constant of the conformationally changed protein can be so strong that it would not elute out of the column (Figure 3). The use of additives may be needed. Arginine prevents binding to the surface [15]. Similarly, a number of other additives may also be used to minimize the secondary interactions. The use of isopropyl alcohol (IPA) as another additive to avoid nonspecific interaction and the reproducibility of the analysis is somewhat discussed in the Section 2.5.

There is no universal protocol for working with additives to avoid nonspecific interactions for all proteins. Chromatographic conditions preventing secondary interactions need to be found out by trial-and-error type of experiments with a variety of additives.

Figure 3. Binding of a protein to the stationary phase and the influence of arginine.

#### 2.3. Effect of particle size and pore size of the stationary phase on SEC separation

Particle size and pore size are important factors for better peak shape, peak sensitivity, and resolution. SEC columns with smaller particle size yield better separation and resolution but with higher back pressure as expected from the basic chromatography theory.

Pore characteristics need to be optimized for SEC separation of biomolecules. To have the high resolution of a mAb monomer from dimer and higher order aggregates as well as from fragments, permeability through the pores of the SEC stationary phases is needed. This is similar to accessibility of the pore needed in reverse phase chromatography (RPC) for better mass transfer kinetics. The larger pore size helps in more efficient permeation of the biomolecules inside the pores, resulting in better size-based separation (Figure 4); 8–14 nm pores are not suitable for the separation of large intact biomolecules and its aggregates since they are too large to enter into the pores. SEC columns with 25 nm pore size are widely available and popular for SEC separation of biomolecules. In the last few years, even larger pore size, such as 30 nm SEC columns, became available in the market, which enables increased permeability of the higher order aggregates of large biomolecules.

Increase in the pore volume of the packing material results into a shallower slope in the calibration curve and increases the spread in elution time between compounds with different molecular masses and improves resolution. But there is a limit to what extent the pore of a particle of definite size can be stretched to its maximum. This is because as the pore volume of a packing material increases, the strength of the packing material generally decreases, making the material more fragile. Figure 5 [16] illustrates the relation between the particle and pore characteristics to the resolution and other peak parameters.

2.4. SEC column selection and calibration curve

standards (globular, branched, and linear).

In the absence of any secondary or non-SEC retention mechanism, the calibration yields an S-shaped curve containing a linear portion in between the total exclusion and total inclusion limits. In Figure 6 [17], the calibration curve was generated using three different types of

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Figure 5. Relation between the particle and pore characteristics to the resolution and other peak parameters.

The red line in Figure 6 represents the average molecular weight of the monoclonal antibody (mAb) IgG (150 kDa). If we follow the calibration curve generated by globular proteins (•), mAb is eluting very close to the total exclusion limit of TSKgel G2000SW, while it is eluting very close to the total inclusion limit of TSKgel G4000SW, resulting into poor separation of the monomer from its impurities in both the cases. But in the middle panel, as seen in the case of TSKgel G3000W, the monomer is eluting around in the middle of the linear range of the calibration curve, so the monomer peak can clearly be separated from its dimer and higher order aggregates and fragment impurities way better than with the other two columns. It is the pore volume between total exclusion and total inclusion volume which is important. The greater the pore volume per unit column volume, the better the separation. In other words, the shallower the calibration curve, the better the separation. All SEC columns with the same dimension, particle size, and pore size from different vendors otherwise may look identical, but the pore volume per unit column volume may not be the same. An analyst can gain

advantage by selecting a column with larger pore volume per unit column volume.

Different SEC columns obtained from different vendors, having the same dimensions and particle size as labeled on the individual columns, may apparently look alike. The difference is in the pore volume per unit column volume due to the differences in particle size and pore size distributions, packing quality, and so on. In general, the better the pore volume per unit

It is important to compromise between the particle sizes and pore volume in order to get the desired separation. Pore characteristics of the SEC column need to be optimized to have a high resolution of a mAb monomer from a dimer and higher order aggregates, as well as from fragments. For large biomolecules, such as therapeutic proteins and monoclonal antibodies (mAbs), a larger exclusion limit will yield better separation particularly of the dimer and higher order aggregates from the monomer.

Figure 4. Permeation of large molecules into pores depends on the pore size.

Figure 5. Relation between the particle and pore characteristics to the resolution and other peak parameters.

#### 2.4. SEC column selection and calibration curve

2.3. Effect of particle size and pore size of the stationary phase on SEC separation

with higher back pressure as expected from the basic chromatography theory.

the higher order aggregates of large biomolecules.

138 Antibody Engineering

characteristics to the resolution and other peak parameters.

Figure 4. Permeation of large molecules into pores depends on the pore size.

higher order aggregates from the monomer.

Particle size and pore size are important factors for better peak shape, peak sensitivity, and resolution. SEC columns with smaller particle size yield better separation and resolution but

Pore characteristics need to be optimized for SEC separation of biomolecules. To have the high resolution of a mAb monomer from dimer and higher order aggregates as well as from fragments, permeability through the pores of the SEC stationary phases is needed. This is similar to accessibility of the pore needed in reverse phase chromatography (RPC) for better mass transfer kinetics. The larger pore size helps in more efficient permeation of the biomolecules inside the pores, resulting in better size-based separation (Figure 4); 8–14 nm pores are not suitable for the separation of large intact biomolecules and its aggregates since they are too large to enter into the pores. SEC columns with 25 nm pore size are widely available and popular for SEC separation of biomolecules. In the last few years, even larger pore size, such as 30 nm SEC columns, became available in the market, which enables increased permeability of

Increase in the pore volume of the packing material results into a shallower slope in the calibration curve and increases the spread in elution time between compounds with different molecular masses and improves resolution. But there is a limit to what extent the pore of a particle of definite size can be stretched to its maximum. This is because as the pore volume of a packing material increases, the strength of the packing material generally decreases, making the material more fragile. Figure 5 [16] illustrates the relation between the particle and pore

It is important to compromise between the particle sizes and pore volume in order to get the desired separation. Pore characteristics of the SEC column need to be optimized to have a high resolution of a mAb monomer from a dimer and higher order aggregates, as well as from fragments. For large biomolecules, such as therapeutic proteins and monoclonal antibodies (mAbs), a larger exclusion limit will yield better separation particularly of the dimer and In the absence of any secondary or non-SEC retention mechanism, the calibration yields an S-shaped curve containing a linear portion in between the total exclusion and total inclusion limits. In Figure 6 [17], the calibration curve was generated using three different types of standards (globular, branched, and linear).

The red line in Figure 6 represents the average molecular weight of the monoclonal antibody (mAb) IgG (150 kDa). If we follow the calibration curve generated by globular proteins (•), mAb is eluting very close to the total exclusion limit of TSKgel G2000SW, while it is eluting very close to the total inclusion limit of TSKgel G4000SW, resulting into poor separation of the monomer from its impurities in both the cases. But in the middle panel, as seen in the case of TSKgel G3000W, the monomer is eluting around in the middle of the linear range of the calibration curve, so the monomer peak can clearly be separated from its dimer and higher order aggregates and fragment impurities way better than with the other two columns. It is the pore volume between total exclusion and total inclusion volume which is important. The greater the pore volume per unit column volume, the better the separation. In other words, the shallower the calibration curve, the better the separation. All SEC columns with the same dimension, particle size, and pore size from different vendors otherwise may look identical, but the pore volume per unit column volume may not be the same. An analyst can gain advantage by selecting a column with larger pore volume per unit column volume.

Different SEC columns obtained from different vendors, having the same dimensions and particle size as labeled on the individual columns, may apparently look alike. The difference is in the pore volume per unit column volume due to the differences in particle size and pore size distributions, packing quality, and so on. In general, the better the pore volume per unit

on protein agitation and temperature-induced aggregation of mAbs has been reported in the literature [18]. Specific racemization of Heavy-Chain Cysteine-220 in the hinge region is identified as a possible cause of degradation during storage. Increased hydrophobicity can increase the likelihood of aggregate formation during manufacturing and storage [19]. Freeze-thaw cycles too can cause an aggregation of an already purified protein [20]. Formation of aggregates may be induced by light as well [21]. That is the reason why all registration applications for new molecular entities and associated drug products require photo-stability data [22]. Increased resolution between the monomer peak and other HMW and LMW impurities is the key for the purification of monoclonal antibodies. SEC can monitor the stability during storage. The removal of HMW and LMW impurities is equally important for application of newly emerging biotherapeutics such as ADCs, bi-specific antibodies, biosimilars, and bio betters as well. As an example from the recent literature report, aggregate and fragment levels were determined by SEC-HPLC for the characterization of bi-specific antibodies [23]. A single chain variable fragment (scFv), composed of the variable regions of the heavy chain (VH) and the light chain (VL), is gaining interest too as it retains the specificity of the original IgG. The scFv format is often used, but one problem that cannot be easily solved by purification is the fact that hybridomas can secrete different monoclonal antibodies [24]. Literature reports a few interesting articles about the concept of mAbs being a perfectly defined entity to researchers. The following is an excerpt from the commentary as shown here [25]. "most researchers consider monoclonal antibodies to be perfectly defined reagents with single specificities" but "Hybridomas frequently secrete more than one light and/or heavy chain." So "the problem is probably best summarized thus: antibodies sold as different are often identical, while antibodies sold as identical are often different (thanks to Natalie de Souza (editor Nature Methods) for this pithy insightful observation), and the customer does not know which is which." In another interesting article, the authors have discussed that "two kappa immunoglobulin light chains are secreted by an anti-DNA hybridoma" and its implications for isotypic exclusion are discussed [26]. Biotherapeutic scientist needs to be aware of these facts while analyzing mAb. The separation of scFvs from dimers and aggregates is also important; most of the affinity

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Different mAbs may have different amount of impurities under native conditions. Representative chromatograms of the separation of four different monoclonal antibodies (IgG1) at 0.75 mL/min using a 4 μm; 4.6 mm ID 15 cm TSKgel SuperSW mAb HTP column are shown in Figure 7 [27]. This analysis clearly showed that under native conditions different monoclonal IgG1 antibodies had different extent of HMW and LMW species based on the individual % peak area analysis (data not shown here). The monomer peak eluted as fast as in 2 min.

A chromatographer needs to choose a suitable column based on the separation criteria the chromatographer is looking for. Comparison of the analysis of mAb aggregates using 15 and 30 cm long TSKgel UP-SW3000, 2 μm columns using the same mobile phase and flow rate is

Fast separation of the HMW and LMW species is important. The effect of the column length should be taken into account when selecting a column in this regard. The results indicate that the TSKgel UP-SW3000 column with a shorter length yielded a similar profile to the 30-cm

purified scFv fractions are monitored by SEC.

shown below [28].

Figure 6. Elution profile of mAb IgG obtained with TSKgel G2000SW, TSKgelG3000W, or TSKgel G4000SW columns.

column volume, the better the separation and resolution of proteins from a SEC column. This is a very critical criterion when selecting a SEC column for better separation.

#### 2.5. Separation of HMW and LMW species by SEC and reproducibility

A chromatographer uses SEC primarily to separate monoclonal antibodies from its impurities or heterogeneities. The monomer IgG1 peak (150 kDa) needs to be purified from its dimer, trimer, or higher order aggregates popularly known as high molecular weight (HMW) species and the fragments which are known as the low molecular weight (LMW) species. SEC is widely accepted as a work horse for routine quality control with its purpose to monitor these HMW and LMW impurities from a mAb monomer. Protein aggregation of biotherapeutics is a common issue. Even a very small amount of aggregates may cause an immunogenic response in the human body and needs to be removed from the monoclonal antibody monomer. More than 99% purity is needed for medicinal purpose [17]. The formulation containing the pure monomer monoclonal antibody needs to be monitored for its stability. The purified protein in the formulation may also undergo aggregation over time. Protein aggregation can happen at any stage during expression and purification. Temperature, pH, ionic strength, concentration and many other factors can give rise to protein instability, leading to aggregation. Effect of ions on protein agitation and temperature-induced aggregation of mAbs has been reported in the literature [18]. Specific racemization of Heavy-Chain Cysteine-220 in the hinge region is identified as a possible cause of degradation during storage. Increased hydrophobicity can increase the likelihood of aggregate formation during manufacturing and storage [19]. Freeze-thaw cycles too can cause an aggregation of an already purified protein [20]. Formation of aggregates may be induced by light as well [21]. That is the reason why all registration applications for new molecular entities and associated drug products require photo-stability data [22]. Increased resolution between the monomer peak and other HMW and LMW impurities is the key for the purification of monoclonal antibodies. SEC can monitor the stability during storage. The removal of HMW and LMW impurities is equally important for application of newly emerging biotherapeutics such as ADCs, bi-specific antibodies, biosimilars, and bio betters as well. As an example from the recent literature report, aggregate and fragment levels were determined by SEC-HPLC for the characterization of bi-specific antibodies [23]. A single chain variable fragment (scFv), composed of the variable regions of the heavy chain (VH) and the light chain (VL), is gaining interest too as it retains the specificity of the original IgG. The scFv format is often used, but one problem that cannot be easily solved by purification is the fact that hybridomas can secrete different monoclonal antibodies [24]. Literature reports a few interesting articles about the concept of mAbs being a perfectly defined entity to researchers. The following is an excerpt from the commentary as shown here [25]. "most researchers consider monoclonal antibodies to be perfectly defined reagents with single specificities" but "Hybridomas frequently secrete more than one light and/or heavy chain." So "the problem is probably best summarized thus: antibodies sold as different are often identical, while antibodies sold as identical are often different (thanks to Natalie de Souza (editor Nature Methods) for this pithy insightful observation), and the customer does not know which is which." In another interesting article, the authors have discussed that "two kappa immunoglobulin light chains are secreted by an anti-DNA hybridoma" and its implications for isotypic exclusion are discussed [26]. Biotherapeutic scientist needs to be aware of these facts while analyzing mAb. The separation of scFvs from dimers and aggregates is also important; most of the affinity purified scFv fractions are monitored by SEC.

Different mAbs may have different amount of impurities under native conditions. Representative chromatograms of the separation of four different monoclonal antibodies (IgG1) at 0.75 mL/min using a 4 μm; 4.6 mm ID 15 cm TSKgel SuperSW mAb HTP column are shown in Figure 7 [27]. This analysis clearly showed that under native conditions different monoclonal IgG1 antibodies had different extent of HMW and LMW species based on the individual % peak area analysis (data not shown here). The monomer peak eluted as fast as in 2 min.

column volume, the better the separation and resolution of proteins from a SEC column. This

Figure 6. Elution profile of mAb IgG obtained with TSKgel G2000SW, TSKgelG3000W, or TSKgel G4000SW columns.

A chromatographer uses SEC primarily to separate monoclonal antibodies from its impurities or heterogeneities. The monomer IgG1 peak (150 kDa) needs to be purified from its dimer, trimer, or higher order aggregates popularly known as high molecular weight (HMW) species and the fragments which are known as the low molecular weight (LMW) species. SEC is widely accepted as a work horse for routine quality control with its purpose to monitor these HMW and LMW impurities from a mAb monomer. Protein aggregation of biotherapeutics is a common issue. Even a very small amount of aggregates may cause an immunogenic response in the human body and needs to be removed from the monoclonal antibody monomer. More than 99% purity is needed for medicinal purpose [17]. The formulation containing the pure monomer monoclonal antibody needs to be monitored for its stability. The purified protein in the formulation may also undergo aggregation over time. Protein aggregation can happen at any stage during expression and purification. Temperature, pH, ionic strength, concentration and many other factors can give rise to protein instability, leading to aggregation. Effect of ions

is a very critical criterion when selecting a SEC column for better separation.

2.5. Separation of HMW and LMW species by SEC and reproducibility

140 Antibody Engineering

A chromatographer needs to choose a suitable column based on the separation criteria the chromatographer is looking for. Comparison of the analysis of mAb aggregates using 15 and 30 cm long TSKgel UP-SW3000, 2 μm columns using the same mobile phase and flow rate is shown below [28].

Fast separation of the HMW and LMW species is important. The effect of the column length should be taken into account when selecting a column in this regard. The results indicate that the TSKgel UP-SW3000 column with a shorter length yielded a similar profile to the 30-cm

Figure 7. Separation of four different mAbs on a 4 μm; 4.6 mm ID 15 cm TSKgel SuperSW mAb HTP column.

column with 50% less run time and 50% lower backpressure at a typical flow rate of 0.35 mL/ min (Figure 8). The resolution between dimer and monomer is still maintained within the acceptable range. Thus, a shorter column could be successfully used for the separation of the dimer and monomer, reducing the overall runtime by half but the resolution of the fragment on the LMW side of the monomer slightly decreased. The longer column yielded a better resolution. As long as the resolution is 1.5 and above yielding a baseline resolution of the two species, the method may remain acceptable. The selection of the correct length of the column should be based on the goal of the separation. The 15-cm column operated at the typical flow rate of 0.35 mL/min yielded a backpressure of 11 Mpa, which was well within its maximum operable pressure and thus could be used in both HPLC and UHPLC systems.

Reproducibility is important in SEC analysis of mAbs. The survey clearly showed the importance of column lifetime and reproducibility for column selection [29, 30]. The survey also shows how the same topic developed over a number of years. Factors which should be

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Two different sources of silica can be a factor in lot-to-lot reproducibility. Vendors always

Bonding chemistry developed on the same silica at different times can also be a factor in lot-to-

lot reproducibility.

considered to select an HPLC column supplier are shown in Table 1.

Table 1. Factors that play a role in selecting a suitable HPLC column.

maintain a strict quality control passing criteria if the silica source is different.

Figure 8. Effect of protein separation on the column length of SEC columns.


Table 1. Factors that play a role in selecting a suitable HPLC column.

column with 50% less run time and 50% lower backpressure at a typical flow rate of 0.35 mL/ min (Figure 8). The resolution between dimer and monomer is still maintained within the acceptable range. Thus, a shorter column could be successfully used for the separation of the dimer and monomer, reducing the overall runtime by half but the resolution of the fragment on the LMW side of the monomer slightly decreased. The longer column yielded a better resolution. As long as the resolution is 1.5 and above yielding a baseline resolution of the two species, the method may remain acceptable. The selection of the correct length of the column should be based on the goal of the separation. The 15-cm column operated at the typical flow rate of 0.35 mL/min yielded a backpressure of 11 Mpa, which was well within its maximum

Figure 7. Separation of four different mAbs on a 4 μm; 4.6 mm ID 15 cm TSKgel SuperSW mAb HTP column.

142 Antibody Engineering

operable pressure and thus could be used in both HPLC and UHPLC systems.

Figure 8. Effect of protein separation on the column length of SEC columns.

Reproducibility is important in SEC analysis of mAbs. The survey clearly showed the importance of column lifetime and reproducibility for column selection [29, 30]. The survey also shows how the same topic developed over a number of years. Factors which should be considered to select an HPLC column supplier are shown in Table 1.

Two different sources of silica can be a factor in lot-to-lot reproducibility. Vendors always maintain a strict quality control passing criteria if the silica source is different.

Bonding chemistry developed on the same silica at different times can also be a factor in lot-tolot reproducibility.

Figure 9 [31] shows the reproducibility of five consecutive injections with low %RSD of all the

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Reproducibility of the analysis of a protein standard mixture with low %RSD of the peak parameters such as retention time, peak area, peak asymmetry and number of theoretical plates and passing the vendor-defined QC criteria can be used for monitoring the column health. Routine users purifying a particular mAb with a particualr SEC column may use the same mAb as their internal standard to monitor the column quality and lifetime over a number of injections. Nowadays, mAb standards from USP and NIST are available for similar purpose. Reproducibility of 15 consecutive injections during the analysis of a USP mAb using a 15-cm TSKgel UP-SW3000 column at 0.5 mL/min flow rate and phosphate buffer at pH 6.7 is shown

The mAb monomer peak eluted at 2.717 min with good resolution between monomer and the dimer peaks as well as the fragments. Similar reproducibility is noticed in case of pH 6.2 (data

Reproducibility in the analysis of the USP mAb in pH 6.2 conditions with 250 mM KCl at 0.3 mL/min using a 30 cm column is shown below—the overlay of the 15 consecutive injections demonstrated consistency (Figure 11) (all USP Reference Standards are provided as delivered and specified by the US Pharmacopeia). The monomer peak elution time was 8.367 min. Similar reproducibility was obtained using a 15-cm column at pH 6.7 (data not shown here). Nonspecific absorption of antibodies onto the column gel matrix poses a challenge, and some newly engineered antibodies possess a high degree of hydrophobicity. The use of organic

Figure 10. Reproducibility of 15 consecutive analytical injections of a USP mAb using a 15-cm TSKgel UP-SW3000

column at phosphate buffer pH 6.7. The overlay of 15 injections is shown.

peak parameters (data not shown here).

in Figure 10.

not shown here).

A robust method with a good SEC analytical column is critical for the analyst. A protein standard mixture can be used to confirm the lot-to-lot reproducibility of an SEC column. The protein standards are chosen to cover the whole range of calibration curve from total exclusion limit to total inclusion limit. A representative chromatogram of the analysis of a protein standard mixture using a TSKgel G3000SWXL, 5 μm, 7.8 mm ID 30 cm column is shown in Figure 9. Thyroglobulin (700 kDa) is close to the total exclusion limit and para-amino-benzoic acid (PABA–137 Da) is near the total inclusion limit. Chromatographers use also vitamin B12 (1.4 kDa) in place of PABA. Vendors generally pass the packed SEC columns using a protein standard mixture and establish a QC pass criteria. For example, the specification for TSKgel G3000SWXL column passing QC is N (PABA) > 20,000 and As (PABA) = (0.7–1.6) where N represents the number of theoretical plates and As represents the peak asymmetry.

Figure 9. Retention time of five different proteins analyzed with a TSKgel G3000SWXL column was high reproducible. An overlay of five injections is shown.

Figure 9 [31] shows the reproducibility of five consecutive injections with low %RSD of all the peak parameters (data not shown here).

Reproducibility of the analysis of a protein standard mixture with low %RSD of the peak parameters such as retention time, peak area, peak asymmetry and number of theoretical plates and passing the vendor-defined QC criteria can be used for monitoring the column health. Routine users purifying a particular mAb with a particualr SEC column may use the same mAb as their internal standard to monitor the column quality and lifetime over a number of injections. Nowadays, mAb standards from USP and NIST are available for similar purpose.

Reproducibility of 15 consecutive injections during the analysis of a USP mAb using a 15-cm TSKgel UP-SW3000 column at 0.5 mL/min flow rate and phosphate buffer at pH 6.7 is shown in Figure 10.

A robust method with a good SEC analytical column is critical for the analyst. A protein standard mixture can be used to confirm the lot-to-lot reproducibility of an SEC column. The protein standards are chosen to cover the whole range of calibration curve from total exclusion limit to total inclusion limit. A representative chromatogram of the analysis of a protein standard mixture using a TSKgel G3000SWXL, 5 μm, 7.8 mm ID 30 cm column is shown in Figure 9. Thyroglobulin (700 kDa) is close to the total exclusion limit and para-amino-benzoic acid (PABA–137 Da) is near the total inclusion limit. Chromatographers use also vitamin B12 (1.4 kDa) in place of PABA. Vendors generally pass the packed SEC columns using a protein standard mixture and establish a QC pass criteria. For example, the specification for TSKgel G3000SWXL column passing QC is N (PABA) > 20,000 and As (PABA) = (0.7–1.6) where N

represents the number of theoretical plates and As represents the peak asymmetry.

Figure 9. Retention time of five different proteins analyzed with a TSKgel G3000SWXL column was high reproducible.

An overlay of five injections is shown.

144 Antibody Engineering

The mAb monomer peak eluted at 2.717 min with good resolution between monomer and the dimer peaks as well as the fragments. Similar reproducibility is noticed in case of pH 6.2 (data not shown here).

Reproducibility in the analysis of the USP mAb in pH 6.2 conditions with 250 mM KCl at 0.3 mL/min using a 30 cm column is shown below—the overlay of the 15 consecutive injections demonstrated consistency (Figure 11) (all USP Reference Standards are provided as delivered and specified by the US Pharmacopeia). The monomer peak elution time was 8.367 min. Similar reproducibility was obtained using a 15-cm column at pH 6.7 (data not shown here).

Nonspecific absorption of antibodies onto the column gel matrix poses a challenge, and some newly engineered antibodies possess a high degree of hydrophobicity. The use of organic

Figure 10. Reproducibility of 15 consecutive analytical injections of a USP mAb using a 15-cm TSKgel UP-SW3000 column at phosphate buffer pH 6.7. The overlay of 15 injections is shown.

Figure 11. Reproducibility of 15 consecutive analytical injections of a USP mAb using a 30-cm TSKgel UP-SW3000 column at pH 6.2. The overlay of 15 injections is shown.

solvents such as isopropyl alcohol (IPA) or salts can decrease this interaction as reported by many scientists. However, the additives may alter the diffusion of these molecules, which results in retention time shift and poor peak resolution that did not occur in a typical aqueous buffer system, such as sodium phosphate buffer at neutral pH. The Figure 12 and Table 2 show that a TSKgel UP-SW3000, 2 μm SEC column was used for analyzing monoclonal antibodies (mAbs) with the addition of 15% IPA in sodium phosphate buffer, pH 6.7. As demonstrated, peak resolution and retention time shift were not impacted.

> column's particle chemistry and packing are optimized so that with the addition of an appropriate amount of selected organic solvents, there is no alteration of peak retention time or poor

> Figure 12. Reproducibility of 14 consecutive analytical injections of a USP mAb using a 30-cm TSKgel UP-SW3000, 2 μm

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Sample load, in both volumetric load and absolute load, may affect SEC separation. Mass overload takes place when sample molecules no longer have free access to diffuse into and out of the pores, thus bypassing part of the column and thereby effectively reducing the length of the column that remains to fractionate the sample. If the height equivalent theoretical plates (HETP) are plotted against the load amount, the HETP values should remain constant as long as the column efficiency is not compromised. The loading capacity is the maximum load

To obtain the capacity of an analytical SEC column as often the chromatographers like to do, a loading study plot HETP vs. load amount is shown above (Figure 14). The loading capacity of a SEC column with defined dimensions depends on the sample. The loading capacity can be

beyond which the HETP value starts increasing, as shown in Figure 14 [33].

SEC column at pH 6.7 with isopropyl alcohol. The overlay of 14 injections is shown.

peak resolution [32].

2.5.1. Loading study

Similarly, Figure 13 shows the overlay of the 15 consecutive injections of USP mAb at pH 6.7 with 15% IPA using a 30-cm column (Figure 13 and Table 3). Improvement in the baseline was noticed after the first two injections. Overall, the analysis yielded excellent reproducibility. The monomer peak elution time was 8.338 min. As expected, there was no considerable difference here compared to the retention time obtained earlier at pH 6.2. The presence of IPA as additive obviously will yield a higher back pressure, and so long as the column is operated within its maximum operable pressure, this should not be an issue. The retention times of monomer, dimer, aggregates, and fragment peaks are nearly unchanged. Peak width and peak shape are very consistent from injection to injection. The baseline of the first injection (as shown in blue) indicate that the column takes only 1–2 injections to be stabilized. After that, all subsequent injections are overlaid perfectly.

The overlay indicates the similarities of peak retention times, peak width and peak height of dimer, monomer, aggregates and fragment peaks between the two different conditions. An appropriate percentage of organic solvent such as isopropyl alcohol (IPA) did not alter the diffusion of mAb molecules using a TSKgel UP-SW3000 column. As demonstrated, this column can be successfully operated with the addition of 15% IPA. Data indicate that the

Figure 12. Reproducibility of 14 consecutive analytical injections of a USP mAb using a 30-cm TSKgel UP-SW3000, 2 μm SEC column at pH 6.7 with isopropyl alcohol. The overlay of 14 injections is shown.

column's particle chemistry and packing are optimized so that with the addition of an appropriate amount of selected organic solvents, there is no alteration of peak retention time or poor peak resolution [32].

#### 2.5.1. Loading study

solvents such as isopropyl alcohol (IPA) or salts can decrease this interaction as reported by many scientists. However, the additives may alter the diffusion of these molecules, which results in retention time shift and poor peak resolution that did not occur in a typical aqueous buffer system, such as sodium phosphate buffer at neutral pH. The Figure 12 and Table 2 show that a TSKgel UP-SW3000, 2 μm SEC column was used for analyzing monoclonal antibodies (mAbs) with the addition of 15% IPA in sodium phosphate buffer, pH 6.7. As

Figure 11. Reproducibility of 15 consecutive analytical injections of a USP mAb using a 30-cm TSKgel UP-SW3000

Similarly, Figure 13 shows the overlay of the 15 consecutive injections of USP mAb at pH 6.7 with 15% IPA using a 30-cm column (Figure 13 and Table 3). Improvement in the baseline was noticed after the first two injections. Overall, the analysis yielded excellent reproducibility. The monomer peak elution time was 8.338 min. As expected, there was no considerable difference here compared to the retention time obtained earlier at pH 6.2. The presence of IPA as additive obviously will yield a higher back pressure, and so long as the column is operated within its maximum operable pressure, this should not be an issue. The retention times of monomer, dimer, aggregates, and fragment peaks are nearly unchanged. Peak width and peak shape are very consistent from injection to injection. The baseline of the first injection (as shown in blue) indicate that the column takes only 1–2 injections to be stabilized. After that, all subsequent

The overlay indicates the similarities of peak retention times, peak width and peak height of dimer, monomer, aggregates and fragment peaks between the two different conditions. An appropriate percentage of organic solvent such as isopropyl alcohol (IPA) did not alter the diffusion of mAb molecules using a TSKgel UP-SW3000 column. As demonstrated, this column can be successfully operated with the addition of 15% IPA. Data indicate that the

demonstrated, peak resolution and retention time shift were not impacted.

injections are overlaid perfectly.

column at pH 6.2. The overlay of 15 injections is shown.

146 Antibody Engineering

Sample load, in both volumetric load and absolute load, may affect SEC separation. Mass overload takes place when sample molecules no longer have free access to diffuse into and out of the pores, thus bypassing part of the column and thereby effectively reducing the length of the column that remains to fractionate the sample. If the height equivalent theoretical plates (HETP) are plotted against the load amount, the HETP values should remain constant as long as the column efficiency is not compromised. The loading capacity is the maximum load beyond which the HETP value starts increasing, as shown in Figure 14 [33].

To obtain the capacity of an analytical SEC column as often the chromatographers like to do, a loading study plot HETP vs. load amount is shown above (Figure 14). The loading capacity of a SEC column with defined dimensions depends on the sample. The loading capacity can be


Table 2. Retention time and peak areas of the monomer and dimer peak (Figure 12).

increased by increasing the column length or diameter. Increasing column length also increases resolution and retention time, leading to an additional separation time and amount of mobile phase.

Below is the loading study of γ-globulin (150 kDa) using the TSKgel UP-SW3000 column. It is necessary to know the experimental range of loading where the retention time, peak shape, separation efficiency, etc., remain nearly unchanged over varying load concentrations. Please note that when a loading study is carried out, both volumetric loading and absolute loading amounts should be studied. In Figure 15, a volumetric loading study is shown. In any SEC analysis, by theory, the total volume injected should not be more than 3% of the column volume to avoid the effect of band broadening.

Peak shape and efficiency were not affected when injecting 400 μg of a monoclonal antibody preparation during a loading study of a monoclonal antibody using a TSKgel G3000SWXL, 5 μm, 7.8 mm ID 30 cm column (Figure 16). A 10-fold increase in total protein content did not affect the retention time, peak symmetry, or separation efficiency of the column [30]. Similar study using a TSKgel G2000SWXL, 5 μm, 7.8 mm ID 30 cm analytical column even with a high load of Bovine Serum Albumin also yielded a well-resolved peak without any splitting [34].

Figure 13. (A) Reproducibility of 15 consecutive analytical injections of a USP mAb using a 30-cm TSKgel UP-SW3000, 2 μm SEC column at pH 6.7 with 15% IPA. The overlay of 15 injections is shown. (B) Chromatogram of 13 a zoomed in.

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As mentioned earlier, having an idea about the sample loading capacity will provide analyst the knowledge about the load range within which the desired sensitivity and resolution can be

Figure 15 shows that even at larger volumetric load containing up to 160 μg proteins, the monomer peak remains well resolved from its dimer. Retention time is remaining constant over the experimental range. Excellent linearity of both monomer peak areas and dimer peak areas versus total load were obtained (data not shown here). So if the primary interest of the analyst is to separate the monomer from the dimer, 160 μg loading in 40 μL volume can be used. Now if the monoclonal antibody concentration can be increased so that 160 μg can be loaded in lower volume (e.g., 10 μL), then the peak shape can further be improved, if needed.

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Figure 13. (A) Reproducibility of 15 consecutive analytical injections of a USP mAb using a 30-cm TSKgel UP-SW3000, 2 μm SEC column at pH 6.7 with 15% IPA. The overlay of 15 injections is shown. (B) Chromatogram of 13 a zoomed in.

increased by increasing the column length or diameter. Increasing column length also increases resolution and retention time, leading to an additional separation time and amount of mobile

Table 2. Retention time and peak areas of the monomer and dimer peak (Figure 12).

Below is the loading study of γ-globulin (150 kDa) using the TSKgel UP-SW3000 column. It is necessary to know the experimental range of loading where the retention time, peak shape, separation efficiency, etc., remain nearly unchanged over varying load concentrations. Please note that when a loading study is carried out, both volumetric loading and absolute loading amounts should be studied. In Figure 15, a volumetric loading study is shown. In any SEC analysis, by theory, the total volume injected should not be more than 3% of the column

Figure 15 shows that even at larger volumetric load containing up to 160 μg proteins, the monomer peak remains well resolved from its dimer. Retention time is remaining constant over the experimental range. Excellent linearity of both monomer peak areas and dimer peak areas versus total load were obtained (data not shown here). So if the primary interest of the analyst is to separate the monomer from the dimer, 160 μg loading in 40 μL volume can be used. Now if the monoclonal antibody concentration can be increased so that 160 μg can be loaded in lower volume (e.g., 10 μL), then the peak shape can further be improved, if needed.

phase.

148 Antibody Engineering

volume to avoid the effect of band broadening.

Peak shape and efficiency were not affected when injecting 400 μg of a monoclonal antibody preparation during a loading study of a monoclonal antibody using a TSKgel G3000SWXL, 5 μm, 7.8 mm ID 30 cm column (Figure 16). A 10-fold increase in total protein content did not affect the retention time, peak symmetry, or separation efficiency of the column [30]. Similar study using a TSKgel G2000SWXL, 5 μm, 7.8 mm ID 30 cm analytical column even with a high load of Bovine Serum Albumin also yielded a well-resolved peak without any splitting [34].

As mentioned earlier, having an idea about the sample loading capacity will provide analyst the knowledge about the load range within which the desired sensitivity and resolution can be


Table 3. Retention time and peak areas of the monomer and dimer peak (Figure 13).

Figure 14. Influence of sample load on height equivalent theoretical plates (HEPT) using three different columns (G2000SW, G3000SW, G4000SW).

achieved. The loading study can be extended to a lower range of detection to get the limit of detection (LOD) and limit of quantitation (LOQ) values for the column in the analysis of mAbs (data not shown here). Similarly, aggregation pattern of mAbs as a function of concentration

Figure 16. Influence of mAb loading on retention time, peak symmetry, or separation efficiency using a TSKgel

Figure 15. (A) Influence of peak resolution and retention time on the amount of γ-globulin loaded on a TSKgel UP-

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SW3000 column; (B) monomer retention time in dependence of the amount of sample.

G3000SWXL, 5 μm, 7.8 mm ID 30 cm column.

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Figure 15. (A) Influence of peak resolution and retention time on the amount of γ-globulin loaded on a TSKgel UP-SW3000 column; (B) monomer retention time in dependence of the amount of sample.

Table 3. Retention time and peak areas of the monomer and dimer peak (Figure 13).

(G2000SW, G3000SW, G4000SW).

150 Antibody Engineering

Figure 14. Influence of sample load on height equivalent theoretical plates (HEPT) using three different columns

Figure 16. Influence of mAb loading on retention time, peak symmetry, or separation efficiency using a TSKgel G3000SWXL, 5 μm, 7.8 mm ID 30 cm column.

achieved. The loading study can be extended to a lower range of detection to get the limit of detection (LOD) and limit of quantitation (LOQ) values for the column in the analysis of mAbs (data not shown here). Similarly, aggregation pattern of mAbs as a function of concentration can be monitored using SEC columns if a mAb is susceptible to aggregation at higher concentration.

#### 2.6. Separation of digestion products of mAbs by size exclusion chromatography

IgG is a relatively large molecule (approx. 150 kDa), and in order to improve the penetration to the tissue, fragmentation is carried out. Digestion with papain or pepsin is commonly applied to obtain antibody fragments without the loss of activity. When papain is used for the antibody digestion, 2 Fab (50 kDa each) and 1 Fc (50 kDa) are obtained from one antibody (Figure 17).

When pepsin is used, a F(ab')2 is obtained. SEC can be used to analyze the separation of these fragments. The scope of this analysis by SEC is taken as an opportunity to explain how to select a column with right particle size, pore size, column dimensions, and so on.

In Figures 18 and 19, a set of four different SEC columns are compared during the separation of papain digestion products of a mAb to explain how to select the right SEC column for the right purpose [35].

For analyzing monoclonal antibody and other biopolymers 250 Å pore size, 5 μm, 7.8 mm ID 30 cm SEC columns are widely considered. For example, TSKgel G3000SWxl columns

> (Panel D) have a separation range for globular protein samples up to 500 kDa. The other SEC column is a 4-μm, 7.8 mm ID 30 cm TSKgel SuperSW mAb HR SEC column (Panel B). It is smaller than the conventional 5 μm TSKgel G3000SWXL column. Smaller particle size and the optimized packing are expected to yield high-resolution analysis of mAb monomers, dimers, and fragments due to shallow calibration slope at the corresponding molar mass region of the mAb monomer (150 kDa). Monomer – dimer resolution increased from 1.63 to 2.02. Another SEC column (Panel A) is a 4-μm TSKgel SuperSW mAb HTP column which is smaller in dimensions, length, and ID (4.6 mm ID 15 cm). This column offers high throughput analysis, separating the dimer and monomer in half the run time compared to all the other three columns in panels B, C, and D. Results are similar to the analysis of mAbs with a 5-μm conventional column (panel D). The fourth column (Panel C) discussed here is of even smaller particle size (3 μm) with higher molar mass exclusion limit (2500 kDa, globular proteins) than all other three columns (500 kDa, globular proteins). Due to the higher exclusion limit, this TSKgel UltraSW aggregate column (Panel C) is expected to yield higher resolution of mAb

> Figure 19. Separation of papain digestion products of a mAb using a set of four different SEC columns. Columns in panel

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A, B, and D are of 250 Å pore sizes, while column in panel D is of 300 Å pore size [35].

multimers and aggregates. Please see further discussion about this in Section 2.7.

Stability of the biotherapeutic proteins in formulation is very critical for candidate selection, characterization of the biotherapeutic, formulation and assay development, and so on. A forced degradation study, popularly known as stress testing, is considerably a faster way to monitor the stability of the therapeutic proteins. Stress is provided by increasing the temperature, changing the pH or a combination of both. Depending on the nature, individual mAbs can be susceptible to light, freeze–thaw conditions, mechanical stress, oxidation and so on. So,

2.7. Forced degradation study by SEC

Figure 17. Cleavage of an IgG with papain or pepsin.


Figure 18. Characteristics of the SEC columns used in the analysis of Figure 19.

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Figure 19. Separation of papain digestion products of a mAb using a set of four different SEC columns. Columns in panel A, B, and D are of 250 Å pore sizes, while column in panel D is of 300 Å pore size [35].

(Panel D) have a separation range for globular protein samples up to 500 kDa. The other SEC column is a 4-μm, 7.8 mm ID 30 cm TSKgel SuperSW mAb HR SEC column (Panel B). It is smaller than the conventional 5 μm TSKgel G3000SWXL column. Smaller particle size and the optimized packing are expected to yield high-resolution analysis of mAb monomers, dimers, and fragments due to shallow calibration slope at the corresponding molar mass region of the mAb monomer (150 kDa). Monomer – dimer resolution increased from 1.63 to 2.02. Another SEC column (Panel A) is a 4-μm TSKgel SuperSW mAb HTP column which is smaller in dimensions, length, and ID (4.6 mm ID 15 cm). This column offers high throughput analysis, separating the dimer and monomer in half the run time compared to all the other three columns in panels B, C, and D. Results are similar to the analysis of mAbs with a 5-μm conventional column (panel D). The fourth column (Panel C) discussed here is of even smaller particle size (3 μm) with higher molar mass exclusion limit (2500 kDa, globular proteins) than all other three columns (500 kDa, globular proteins). Due to the higher exclusion limit, this TSKgel UltraSW aggregate column (Panel C) is expected to yield higher resolution of mAb multimers and aggregates. Please see further discussion about this in Section 2.7.

#### 2.7. Forced degradation study by SEC

can be monitored using SEC columns if a mAb is susceptible to aggregation at higher concen-

IgG is a relatively large molecule (approx. 150 kDa), and in order to improve the penetration to the tissue, fragmentation is carried out. Digestion with papain or pepsin is commonly applied to obtain antibody fragments without the loss of activity. When papain is used for the antibody digestion, 2 Fab (50 kDa each) and 1 Fc (50 kDa) are obtained from one antibody (Figure 17). When pepsin is used, a F(ab')2 is obtained. SEC can be used to analyze the separation of these fragments. The scope of this analysis by SEC is taken as an opportunity to explain how to

In Figures 18 and 19, a set of four different SEC columns are compared during the separation of papain digestion products of a mAb to explain how to select the right SEC column for the

For analyzing monoclonal antibody and other biopolymers 250 Å pore size, 5 μm, 7.8 mm ID 30 cm SEC columns are widely considered. For example, TSKgel G3000SWxl columns

2.6. Separation of digestion products of mAbs by size exclusion chromatography

select a column with right particle size, pore size, column dimensions, and so on.

tration.

152 Antibody Engineering

right purpose [35].

Figure 17. Cleavage of an IgG with papain or pepsin.

Figure 18. Characteristics of the SEC columns used in the analysis of Figure 19.

Stability of the biotherapeutic proteins in formulation is very critical for candidate selection, characterization of the biotherapeutic, formulation and assay development, and so on. A forced degradation study, popularly known as stress testing, is considerably a faster way to monitor the stability of the therapeutic proteins. Stress is provided by increasing the temperature, changing the pH or a combination of both. Depending on the nature, individual mAbs can be susceptible to light, freeze–thaw conditions, mechanical stress, oxidation and so on. So,

monitoring the stability from time to time is important. Literature reports: "Whereas stabilitytesting requirements are defined in regulatory guidelines, standard procedures for forced degradation of therapeutic proteins are largely unavailable, except for photo stability" [36]. Stress conditions induce unfolding of the native protein structure. As a result, the exposed hydrophobic patches may be able to interact with each other, leading to aggregation [37]. Protein degradation can also be measured using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), and the protein structure can be characterized by other techniques using Fourier transform infrared spectrometry and circular dichroism [38]. SEC is widely accepted as a great tool to monitor the forced degradation. Forced degradation by acid denaturation and heat denaturation analyzed using a TSKgel UltraSW Aggregate, 3 μm, 30 nm, 7.8 mm ID 30 cm column is used as an example below [39] (Figure 20).

Acid denaturation: After reducing the pH of the IgG1 sample solution down to 4.7 by adding phosphoric acid, aliquots were analyzed at 5, 20, and 50 min, and the response was compared to that of the original sample solution. The blue trace shows the intact mAb and what is (presumably) its dimer eluting at 8.65 min when analyzed at flow rate of 1 mL/min. The degradation of the monoclonal antibody creates a larger MW entity (unidentified) that elutes directly after the dimer and before the monomer. Continued decay led to increase of both peaks. Clearly the dimer increases in size, while the peak height of the monomer decreases. Hints of higher order "multimers" are detected between 7 and 8 min [31].

The analysis of a heat denatured, large hydrophobic metalloprotein, Apo ferritin, analyzed using a TSKgel UltraSW Aggregate column yielded high resolution between the monomer (450 kDa) and dimer (900 kDa) (Figure 21). The trimer (1350 kDa), tetramer (1800 kDa), and higher order aggregates of Apo ferritin were well separated. Tetramer of Apo ferritin is approximately equivalent to 13 mer of a mAb. Larger exclusion limit yielded better resolution

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Figure 21. Analysis of Apo ferritin after heat denaturation using a TSKgel UltraSW aggregate column.

3. Effect of mobile phase additives on the separation of monoclonal

The use of organic solvents such as isopropyl alcohol (IPA) or salts can decrease the secondary interaction as reported by many scientists and mentioned earlier briefly in this chapter in Section 2.5. Peak resolution and retention time shift were not impacted with the use of 15% IPA as demonstrated in the two examples (Figure 12 and Figure 13). It is necessary to evaluate the individual SEC column regarding the impact of the additives since a stationary phase with minimum impact is always a favorable choice. After important mobile phase parameters have been set, such as pH, stationary phase, and ionic strength, significant improvements can, in fact, be made to separate mAb monomers from aggregates and fragments. There are no universal additives which can be applied for every mAb or protein purification. Other common additives are methanol and ethanol. Use of sodium perchlorate may also improve the separation and resolution. By switching from 0.2 mol/L sodium chloride to 0.2 mol/L of the more chaotropic sodium perchlorate salt, together with a twofold reduction in the buffer concentration, less peak tailing and distinct peaks for the dimer and trimer could be noticed [41]. Sodium dodecyl sulfate (SDS), urea, guanidine hydrochloride, etc., are sometimes used when the proteins need to be solubilized, leading to denaturation of the protein and breakage of the noncovalent bonds. The use of additives should be considered only when needed. In many cases, the performance of the column is irreversibly changed when a column is subjected

of the higher order aggregates. [40].

antibodies

Heat denaturation: Degradation of mAb at pH 5.5 and a temperature of 60C were monitored. Fifty microliters of antibody in 0.1 M phosphate buffer (pH 6.0) was mixed with 50 μL of 0.1 mol/L phosphate buffer, pH 4.65; the final pH was 5.5; and 20 μL was injected [31]. Heating for 1 h at 60C results in almost complete breakdown of the monoclonal antibody and the formation of very large aggregates (multimers) that extend to the exclusion volume of the column. The intensity of the multimer peaks increased as a function of the incubation period at 60C. The larger the total exclusion volume of the column, the better the resolution of higher order aggregates. An SEC column with a 500-kDa exclusion limit may not be able to resolve the individual multimers such as trimers and tetramers. An SEC column with larger exclusion limit, for example, 2500 kDa, may be useful in such a case [31].

Figure 20. Analysis of forced degradation of IgG after acid and heat denaturation using a SEC TSKgel UltraSW aggregate, 3 μm, 30 nm, 7.8 mm ID 30 cm column.

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Figure 21. Analysis of Apo ferritin after heat denaturation using a TSKgel UltraSW aggregate column.

monitoring the stability from time to time is important. Literature reports: "Whereas stabilitytesting requirements are defined in regulatory guidelines, standard procedures for forced degradation of therapeutic proteins are largely unavailable, except for photo stability" [36]. Stress conditions induce unfolding of the native protein structure. As a result, the exposed hydrophobic patches may be able to interact with each other, leading to aggregation [37]. Protein degradation can also be measured using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), and the protein structure can be characterized by other techniques using Fourier transform infrared spectrometry and circular dichroism [38]. SEC is widely accepted as a great tool to monitor the forced degradation. Forced degradation by acid denaturation and heat denaturation analyzed using a TSKgel UltraSW Aggregate, 3 μm,

Acid denaturation: After reducing the pH of the IgG1 sample solution down to 4.7 by adding phosphoric acid, aliquots were analyzed at 5, 20, and 50 min, and the response was compared to that of the original sample solution. The blue trace shows the intact mAb and what is (presumably) its dimer eluting at 8.65 min when analyzed at flow rate of 1 mL/min. The degradation of the monoclonal antibody creates a larger MW entity (unidentified) that elutes directly after the dimer and before the monomer. Continued decay led to increase of both peaks. Clearly the dimer increases in size, while the peak height of the monomer decreases.

Heat denaturation: Degradation of mAb at pH 5.5 and a temperature of 60C were monitored. Fifty microliters of antibody in 0.1 M phosphate buffer (pH 6.0) was mixed with 50 μL of 0.1 mol/L phosphate buffer, pH 4.65; the final pH was 5.5; and 20 μL was injected [31]. Heating for 1 h at 60C results in almost complete breakdown of the monoclonal antibody and the formation of very large aggregates (multimers) that extend to the exclusion volume of the column. The intensity of the multimer peaks increased as a function of the incubation period at 60C. The larger the total exclusion volume of the column, the better the resolution of higher order aggregates. An SEC column with a 500-kDa exclusion limit may not be able to resolve the individual multimers such as trimers and tetramers. An SEC column with larger exclusion

Figure 20. Analysis of forced degradation of IgG after acid and heat denaturation using a SEC TSKgel UltraSW aggre-

30 nm, 7.8 mm ID 30 cm column is used as an example below [39] (Figure 20).

Hints of higher order "multimers" are detected between 7 and 8 min [31].

limit, for example, 2500 kDa, may be useful in such a case [31].

gate, 3 μm, 30 nm, 7.8 mm ID 30 cm column.

154 Antibody Engineering

The analysis of a heat denatured, large hydrophobic metalloprotein, Apo ferritin, analyzed using a TSKgel UltraSW Aggregate column yielded high resolution between the monomer (450 kDa) and dimer (900 kDa) (Figure 21). The trimer (1350 kDa), tetramer (1800 kDa), and higher order aggregates of Apo ferritin were well separated. Tetramer of Apo ferritin is approximately equivalent to 13 mer of a mAb. Larger exclusion limit yielded better resolution of the higher order aggregates. [40].
