**6.1 Salt concentration**

Salts affect the separation of mAb on TSKgel FcR-IIIA-NPR. To best control the pH in the linear gradient, mobile phase should consist of a buffer with suitable buffering capacity whereas neutral salts are used to increase the ionic strength. Both components affect the binding affinity. Buffer provides pH control and salt ions provide charge shielding or stoichiometric ion bonding on the stationary phase and mAbs. Salts impart specific or non-specific effects by modulating protein–protein and protein-surface interactions. Binding affinity to the column depends on the binding constant. Increasing salt concentration have shown to lead to the elution at earlier retention time (**Figure 15**, Panel A), although the intensity of the effect probably is also related to the individual mAb studied. The binding strength is also dependent on the buffer used such as sodium citrate or sodium acetate. Citrate yielded stronger binding and hence higher retention time. Acetate buffer instead yielded better resolution of the peaks as compared to citrate (**Figure 15**, Panel B).

In the affinity chromatography, the optimum flow rate of elution may be dependent on the molecule-specific interaction with the ligand. Irrespective of the flow rates (**Figure 16**), all the three glycoform peaks eluted within 67% of mobile phase B when the analysis was carried out using a linear gradient of 50 mM sodium citrate buffer from pH 6.5 to 4.5 over 50 minutes at 20°C. Although flow rate did not have effect on elution pH, lower flow rate may be used to increase the sensitivity due to longer residence time in the flow cell.

#### **Figure 15.**

*Effect of salt (Panel A) and buffer (Panel B) on the separation of mAb glycoforms.*

**93**

**Figure 17.**

*Effect of gradient slope on the separation efficiency of peaks.*

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

The gradient elution method is common for the separation of species of different binding strength. **Figure 17** shows the effect of a gradient slope on the separation mAb on TSKgel FcR-IIIA-NPR. As expected, longer gradient time increased the resolution between the peaks whereas the overall peak area and relative ratio of the peak areas remained unchanged. **Figure 17** indicates how shallower slope increased the resolution between the three peaks. This is particularly noticeable between peaks 2 and 3. On the other hand, it should be noted that, for any further analytical

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

**6.2 Gradient slope**

**Figure 16.** *Increased sensitivity at lower flow rate.*

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

### **6.2 Gradient slope**

*Monoclonal Antibodies*

**6.1 Salt concentration**

longer residence time in the flow cell.

*Effect of salt (Panel A) and buffer (Panel B) on the separation of mAb glycoforms.*

**6. Factors affecting the chromatographic separation**

Salts affect the separation of mAb on TSKgel FcR-IIIA-NPR. To best control the pH in the linear gradient, mobile phase should consist of a buffer with suitable buffering capacity whereas neutral salts are used to increase the ionic strength. Both components affect the binding affinity. Buffer provides pH control and salt ions provide charge shielding or stoichiometric ion bonding on the stationary phase and mAbs. Salts impart specific or non-specific effects by modulating protein–protein and protein-surface interactions. Binding affinity to the column depends on the binding constant. Increasing salt concentration have shown to lead to the elution at earlier retention time (**Figure 15**, Panel A), although the intensity of the effect probably is also related to the individual mAb studied. The binding strength is also dependent on the buffer used such as sodium citrate or sodium acetate. Citrate yielded stronger binding and hence higher retention time. Acetate buffer instead yielded better resolution of the peaks as compared to citrate (**Figure 15**, Panel B). In the affinity chromatography, the optimum flow rate of elution may be dependent on the molecule-specific interaction with the ligand. Irrespective of the flow rates (**Figure 16**), all the three glycoform peaks eluted within 67% of mobile phase B when the analysis was carried out using a linear gradient of 50 mM sodium citrate buffer from pH 6.5 to 4.5 over 50 minutes at 20°C. Although flow rate did not have effect on elution pH, lower flow rate may be used to increase the sensitivity due to

**92**

**Figure 16.**

**Figure 15.**

*Increased sensitivity at lower flow rate.*

The gradient elution method is common for the separation of species of different binding strength. **Figure 17** shows the effect of a gradient slope on the separation mAb on TSKgel FcR-IIIA-NPR. As expected, longer gradient time increased the resolution between the peaks whereas the overall peak area and relative ratio of the peak areas remained unchanged. **Figure 17** indicates how shallower slope increased the resolution between the three peaks. This is particularly noticeable between peaks 2 and 3. On the other hand, it should be noted that, for any further analytical

**Figure 17.** *Effect of gradient slope on the separation efficiency of peaks.*

work (e.g. for mass spec), longer gradient increases the peak volumes and thus peak fractions will be more diluted.

## **6.3 Temperature**

The ligand in FcR-IIIA-NPR column is a 20-kDa folded polypeptide and thus special care is to be considered to maintain protein conformation intact with proper run temperature. **Figure 18** shows the separation of mAb at four different temperatures (5°C, 15°C, 20°C, 25°C) at flow rate of 0.2 mL/min. As the temperature increased, the retention time of the three peaks decreased, indicating somewhat lower binding affinity as a function of higher temperature. However, importantly, overall peak profile at each temperature remains unchanged. Thus, for practical reasons, temperature range from 15 to 25°C is recommended for most analytical work. Following the completion of the analysis the column needs to be stored at 2–8°C.

## **6.4 Sample load**

**Figure 19** shows the effect of load amount of mAb on the separation profile. The limit of detection was determined as 1.5 μg as per USP definition S/N of 2–3. A load of 3.16 μg could still be easily quantified (LOQ ). The analysis was repeatable, robust and the total peak area increased proportionately as the load amount was

**Figure 18.** *Effect of temperature on the separation of mAb.*

**95**

**7.1 Selectivity**

thus elutes in void volume.

**7.2 Lot-to-lot variability**

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

for the best resolution and for maintaining the lifetime of the column.

for mAbs containing any amount of aggregates or oxidized forms.

**7. Robustness of TSKgel FcR-IIIA-NPR affinity column**

The usefulness of any affinity chromatography column depends on several robustness factors. Here, selectivity is dependent on the nature of N-glycan. This is clearly demonstrated by analyzing enzymatically deglycosylated mAb. PNGase-F deglycosidase reacts between asparagine residue and the innermost N-acetyl glucosamine (GlcNAc) of the complex oligosaccharide or high mannose content. **Figure 20** shows that enzymatically deglycosylated NIST mAb does not bind to the column and

Scope of quality control of the therapeutic antibodies is expanding rapidly due to the emergence of biosimilars, "biobetter" forms and numerous other kinds of biologics in the biotherapeutic market. Lot-to-lot difference in the activity of innovator mAb may vary up to 20% in the manufacturing process [25]. Although substantial improvement has been attained in CHO cell engineering during recent years, and different strategies are there e.g., to produce afucosylated antibody drugs, still not enough technology is available to fully control *in vivo* glycosylation during production [26]. The lot-to-lot difference in N-glycan content may give rise to a wide variety of risk and thus N-glycan heterogeneity is a key factor to be monitored in quality control. To demonstrate importance of the mAb lot-to-lot quality control, two manufacturing lots of mAb were analyzed using the TSKgel FcR-IIIA-NPR column (**Figure 21**). Both lots yielded a similar 3-peak elution profile. However, when percentual peak areas of the individual peaks were compared to check the consistency between the two mAb lots. Lot B showed a higher glycan percentage in peak

increased in a linear manner in consecutive injections. Relative ratio of the individual peak areas in the three peaks remained constant. The column can generally be used up to 100 μg protein load. However, 5–50 μg load of mAb is recommended

Presence of aggregates in IgG samples impact the binding to the Fcγ receptors. A recently published article reports that deamidated IgG samples caused aggregation or formation of higher molecular weight (HMW) species and thereby impacted the binding affinity. Asparagine deamidation led to reduced binding of IgG to the low affinity FcγRIIIa receptor [24]. IgGs may also be more prone to aggregation when glycans are absent, which in turn has an effect on Fc effector functions. Lack of glycan and its effect on binding is explained below in Section 7.1. IgG dimers and aggregates may also bind stronger to different types of Fc receptors and thus have significant impact on affinity determination. Accumulated strength of multiple non-covalent affinities between the ligand and the receptor is known as avidity effect. This effect can alter the binding to the receptor and should be considered during the analysis mAb with dimer and higher order aggregates. The interaction, if any, needs to be evaluated in case-by-case basis. Up to 5% of aggregates in IgG samples changed the binding and kinetics to each of the Fc receptors [24]. Methionine is easily oxidized to methionine sulfoxide which can also lead to light chain aggregation. Oxidation has impact on the binding to the Fcγ receptors and depends on the extent of oxidation. As reported [24], methionine oxidation below 7% did not impact on binding to the receptors. Taken together, all above factors should be considered when using this column, especially during analysis method development

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

**Figure 19.** *Effect of sample load on the separation of mAb.*

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

increased in a linear manner in consecutive injections. Relative ratio of the individual peak areas in the three peaks remained constant. The column can generally be used up to 100 μg protein load. However, 5–50 μg load of mAb is recommended for the best resolution and for maintaining the lifetime of the column.

Presence of aggregates in IgG samples impact the binding to the Fcγ receptors. A recently published article reports that deamidated IgG samples caused aggregation or formation of higher molecular weight (HMW) species and thereby impacted the binding affinity. Asparagine deamidation led to reduced binding of IgG to the low affinity FcγRIIIa receptor [24]. IgGs may also be more prone to aggregation when glycans are absent, which in turn has an effect on Fc effector functions. Lack of glycan and its effect on binding is explained below in Section 7.1. IgG dimers and aggregates may also bind stronger to different types of Fc receptors and thus have significant impact on affinity determination. Accumulated strength of multiple non-covalent affinities between the ligand and the receptor is known as avidity effect. This effect can alter the binding to the receptor and should be considered during the analysis mAb with dimer and higher order aggregates. The interaction, if any, needs to be evaluated in case-by-case basis. Up to 5% of aggregates in IgG samples changed the binding and kinetics to each of the Fc receptors [24]. Methionine is easily oxidized to methionine sulfoxide which can also lead to light chain aggregation. Oxidation has impact on the binding to the Fcγ receptors and depends on the extent of oxidation. As reported [24], methionine oxidation below 7% did not impact on binding to the receptors. Taken together, all above factors should be considered when using this column, especially during analysis method development for mAbs containing any amount of aggregates or oxidized forms.

#### **7. Robustness of TSKgel FcR-IIIA-NPR affinity column**

#### **7.1 Selectivity**

*Monoclonal Antibodies*

**6.3 Temperature**

**6.4 Sample load**

fractions will be more diluted.

work (e.g. for mass spec), longer gradient increases the peak volumes and thus peak

The ligand in FcR-IIIA-NPR column is a 20-kDa folded polypeptide and thus special care is to be considered to maintain protein conformation intact with proper run temperature. **Figure 18** shows the separation of mAb at four different temperatures (5°C, 15°C, 20°C, 25°C) at flow rate of 0.2 mL/min. As the temperature increased, the retention time of the three peaks decreased, indicating somewhat lower binding affinity as a function of higher temperature. However, importantly, overall peak profile at each temperature remains unchanged. Thus, for practical reasons, temperature range from 15 to 25°C is recommended for most analytical work. Following

**Figure 19** shows the effect of load amount of mAb on the separation profile. The limit of detection was determined as 1.5 μg as per USP definition S/N of 2–3. A load of 3.16 μg could still be easily quantified (LOQ ). The analysis was repeatable, robust and the total peak area increased proportionately as the load amount was

the completion of the analysis the column needs to be stored at 2–8°C.

**94**

**Figure 19.**

**Figure 18.**

*Effect of temperature on the separation of mAb.*

*Effect of sample load on the separation of mAb.*

The usefulness of any affinity chromatography column depends on several robustness factors. Here, selectivity is dependent on the nature of N-glycan. This is clearly demonstrated by analyzing enzymatically deglycosylated mAb. PNGase-F deglycosidase reacts between asparagine residue and the innermost N-acetyl glucosamine (GlcNAc) of the complex oligosaccharide or high mannose content. **Figure 20** shows that enzymatically deglycosylated NIST mAb does not bind to the column and thus elutes in void volume.

#### **7.2 Lot-to-lot variability**

Scope of quality control of the therapeutic antibodies is expanding rapidly due to the emergence of biosimilars, "biobetter" forms and numerous other kinds of biologics in the biotherapeutic market. Lot-to-lot difference in the activity of innovator mAb may vary up to 20% in the manufacturing process [25]. Although substantial improvement has been attained in CHO cell engineering during recent years, and different strategies are there e.g., to produce afucosylated antibody drugs, still not enough technology is available to fully control *in vivo* glycosylation during production [26]. The lot-to-lot difference in N-glycan content may give rise to a wide variety of risk and thus N-glycan heterogeneity is a key factor to be monitored in quality control.

To demonstrate importance of the mAb lot-to-lot quality control, two manufacturing lots of mAb were analyzed using the TSKgel FcR-IIIA-NPR column (**Figure 21**). Both lots yielded a similar 3-peak elution profile. However, when percentual peak areas of the individual peaks were compared to check the consistency between the two mAb lots. Lot B showed a higher glycan percentage in peak

**Figure 20.** *Deglycosylated mAb does not bind to the column.*

**Figure 21.**

*N-glycan analysis of two manufacturing lots of a therapeutic antibody on TSKgel FcR-IIIA-NPR.*

1 (42% versus 34%) and lower percentual amounts in peaks 2 and 3. In a subsequent ADCC assay, this also correlated with lower ADCC activity in the lot B. This experiment thus supports the notion that FcR affinity chromatography is suitable for lot-to-lot quality control of therapeutic mAbs.

To confirm consistency in FcR column manufacturing is also equally important for quality control. Three different lots of TSKgel FcR-IIIA-NPR column (Lots A, B, and C) were tested using reference mAb sample under identical chromatographic conditions (**Figure 22**). No significant variation in 3-peak profile was noticed between the three different column lots.

#### **7.3 Effect of host cell proteins on the separation of mAb**

Most mAb pharmaceuticals are produced in CHO cell culture system. Host cell proteins (HCPs), or host cell impurities, are collectively recognized as several forms of host cell products such as DNA, proteins, endotoxin and, if contaminated, viruses. These together are considered as process-related contaminants. They often have antigenic or pyrogenic effects in human and thus must be removed during downstream processing.

**97**

**Figure 23.**

**Figure 22.**

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

With regard to QC characterization, it is also necessary to assess if host cell proteins can interfere mAb binding on a TSKgel FcR-IIIA-NPR column. In the following study, CHO cell culture supernatant ("feedstock") was directly used for FcR column analysis and the results were compared to a previously purified mAb in the same assay (**Figure 23**, Panel a). However, no significant difference was noticed between the two profiles. This indicates that the HCPs had no significant impact on the mAb affinity to FcR column and about 5 μg of mAb in a feedstock was enough to obtain a suitable signal for monitoring process development in a bioreactor. The robustness of the assay was further tested using the mAb in CHO cell supernatant with 200 consecutive injections (**Figure 23**, Panel b). The total peak area remained constant with a % RSD (n = 10) of 0.79. It was also noticed that addition of NaCl to minimize unwanted non-specific interactions further improved durability at 20°C. The **Figure 23** (Panel c) shows how FcR column can be used for cell line selection and upstream monitoring. In this case, samples from CHO cell culture supernatant were collected, filtered, captured on protein A, and then injected to a TSKgel FcR-IIIA-NPR column. NaCl was added to improve separation. Glycoform changes in mAb were monitored over 14 days. The proportion of the intensities and peak areas of the three peaks significantly changed over the days that can be correlated to indicate changes in ADCC activity during

*(a) Analysis of CHO cell feedstock containing mAb versus purified mAb. (b) Assessment of FcR column stability over 200 injections. (c) Monitoring of glycan composition changes during fermentation.*

*Lot-to-lot consistency in TSKgel FcR-IIIA-NPR column manufacturing.*

*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*

With regard to QC characterization, it is also necessary to assess if host cell proteins can interfere mAb binding on a TSKgel FcR-IIIA-NPR column. In the following study, CHO cell culture supernatant ("feedstock") was directly used for FcR column analysis and the results were compared to a previously purified mAb in the same assay (**Figure 23**, Panel a). However, no significant difference was noticed between the two profiles. This indicates that the HCPs had no significant impact on the mAb affinity to FcR column and about 5 μg of mAb in a feedstock was enough to obtain a suitable signal for monitoring process development in a bioreactor. The robustness of the assay was further tested using the mAb in CHO cell supernatant with 200 consecutive injections (**Figure 23**, Panel b). The total peak area remained constant with a % RSD (n = 10) of 0.79. It was also noticed that addition of NaCl to minimize unwanted non-specific interactions further improved durability at 20°C. The **Figure 23** (Panel c) shows how FcR column can be used for cell line selection and upstream monitoring. In this case, samples from CHO cell culture supernatant were collected, filtered, captured on protein A, and then injected to a TSKgel FcR-IIIA-NPR column. NaCl was added to improve separation. Glycoform changes in mAb were monitored over 14 days. The proportion of the intensities and peak areas of the three peaks significantly changed over the days that can be correlated to indicate changes in ADCC activity during

**Figure 22.** *Lot-to-lot consistency in TSKgel FcR-IIIA-NPR column manufacturing.*

#### **Figure 23.**

*(a) Analysis of CHO cell feedstock containing mAb versus purified mAb. (b) Assessment of FcR column stability over 200 injections. (c) Monitoring of glycan composition changes during fermentation.*

*Monoclonal Antibodies*

**96**

**Figure 21.**

**Figure 20.**

*Deglycosylated mAb does not bind to the column.*

downstream processing.

1 (42% versus 34%) and lower percentual amounts in peaks 2 and 3. In a subsequent ADCC assay, this also correlated with lower ADCC activity in the lot B. This experiment thus supports the notion that FcR affinity chromatography is suitable

*N-glycan analysis of two manufacturing lots of a therapeutic antibody on TSKgel FcR-IIIA-NPR.*

To confirm consistency in FcR column manufacturing is also equally important for quality control. Three different lots of TSKgel FcR-IIIA-NPR column (Lots A, B, and C) were tested using reference mAb sample under identical chromatographic conditions (**Figure 22**). No significant variation in 3-peak profile was noticed

Most mAb pharmaceuticals are produced in CHO cell culture system. Host cell proteins (HCPs), or host cell impurities, are collectively recognized as several forms of host cell products such as DNA, proteins, endotoxin and, if contaminated, viruses. These together are considered as process-related contaminants. They often have antigenic or pyrogenic effects in human and thus must be removed during

for lot-to-lot quality control of therapeutic mAbs.

**7.3 Effect of host cell proteins on the separation of mAb**

between the three different column lots.

**Figure 24.** *Acid stability of a recombinant Fc*γ*RIII ligand as compared to a wild type ligand***.**

fermentation. Thus, by monitoring samples from the bioreactor using TSKgel FcR-IIIA-NPR, process engineers can approximate the optimal day for desired yield and ADCC activity.

#### **7.4 Column pH stability and cleaning**

Recommended working pH for the FcR column as mentioned in operational conditions and specifications (OCS) is from pH 8 to 4. As mentioned earlier, the protein ligand contains eight amino acid substitutions for improved stability. To further test acid stability, the column was held at pH3 for 200 hours. The modified ligand did not lose its binding affinity and the selectivity while the wild-type lost the binding affinity and selectivity within one hour (**Figure 24**). Based on this and other studies, a pH range of 3–8 is can be used for short term and pH 4.5–7 for long term usage. Due to a protein nature of the ligand, acetonitrile and other organic solvents are not suitable for the column. For cleaning, 3–5 injections of 0.5–2 mL of a buffer containing 500 mM NaCl or 20% ethanol can safely be used in reverse direction at half the normal flow rate. Once the cleaning procedure is complete, it is necessary to equilibrate the column in mobile phase for at least 45 minutes. Cleaning with alkalic solutions above pH 8 are not recommended since this will denature the protein ligand. Sodium azide (0.05%) can be used in the mobile phase as antibacterial agent.

## **8. Mass spectrometric characterization of glycoform peaks separated by TSKgel FcR-IIIA-NPR column**

Mass spectrometric characterization is becoming an integral part of the liquid chromatography analysis. As an example how TSKgel FcR-IIIA-NPR column can be utilized in mass spec work, we describe here an in-line LC–MS intact mAb analysis of trastuzumab (Herceptin Biosimilar). The analysis was carried out using 100 mM volatile ammonium acetate buffer and a linear pH gradient from pH 6.5 to pH 4.5 at the flow rate of 0.4 mL/min. The wavelenght of detection was 280 nm. The column temperature during the analysis was maintained at 20°C. Three glycoform peaks could be detected by UV detector. Mass spectrometric detection was carried out using SCIEX X500B Q-TOF in ESI positive mode, within mass/charge (m/z) range of 5000–7000. Ion source gases 1 and 2 were maintained at 50 psi, curtain gas at 30 psi, CAD gas at 7 psi and temperature at 450°C. Spray voltage was maintained at 5200 V, declustering

**99**

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

potential at 275 V, and collison energy at 20 V. Time bins to sum was set at 120. For the automated characterization of the data acquired on the X500B QTOF, SCIEX Biotool kit software was used. Total Ion Chromatogram (TIC) was obtained by summing up intensities of all mass spectral peaks belonging to the same scan. An overlay of UV profile and TIC profile is shown in **Figure 25**. For further analysis of glyosylation profiles

As mentioned earlier, organic solvents such as acetonitrile are not suitable for the column and vapor pressure of water is very low. Thus, volatile salts such as 100 mM ammonium acetate or ammonium formate are used. To avoid ion source contamination during prolonged use, molarity should be kept at low (preferably <50 mM). Depending on the need for further optimization for different mAbs, volatile salt ammonium bicarbonate can also be used as such or in combination with other volatile salts.

The C-terminal part of the heavy chains contains the Fc fragment which is responsible for cellular effector functions, essential for proper function of most therapeutic mAbs. In some cases, it is desirable to express fragment antibodies that are smaller than intact mAbs but still are capable of eliciting their therapeutic function by activation of the immune system. Literature reports that both glycoengineering and protein engineering have rendered Fc domains with enhanced Fc receptor binding. In general, Fc fragments and their numerous variants are rapidly gaining

The binding efficiency of the Fc fragment was tested in-house to assess suitability of TSKgel FcR-IIIA-NPR column on the characterization of smaller fragment antibodies. In short, trastuzumab was fragmented with papain that cleaves IgG at His228 forming two Fab parts and one Fc part (**Figure 26**). The reaction mixture was incubated at 37°C for 15 minutes to activate papain followed by mAb addition and further incubation overnight at room temperature. Papain activity was stopped with 5 mM iodoacetamide. A control a sample (no papain during incubation) and a sample from papain digestion were used for this study. Size exclusion chromatography followed by mass spectrometric analysis confirmed >95% cleavage of mAb to Fc

Both the control mAb and fragments were analyzed on TSKgel FcR-IIIA-NPR column. As expected, Fab did not bind to the FcR column but eluted in

of these three peaks, SCIEX BioPharmaView™ software can be used.

*Overlay of UV spectrum and Total Ion Chromatogram (TIC) of Herceptin Biosimilar.*

**9. Analysis of Fc fragment on TSKgel FcR-IIIA-NPR**

interest as a platform in the development of efficient biotherapeutics.

and Fab fragments (data available by request).

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

**Figure 25.**

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

**Figure 25.** *Overlay of UV spectrum and Total Ion Chromatogram (TIC) of Herceptin Biosimilar.*

potential at 275 V, and collison energy at 20 V. Time bins to sum was set at 120. For the automated characterization of the data acquired on the X500B QTOF, SCIEX Biotool kit software was used. Total Ion Chromatogram (TIC) was obtained by summing up intensities of all mass spectral peaks belonging to the same scan. An overlay of UV profile and TIC profile is shown in **Figure 25**. For further analysis of glyosylation profiles of these three peaks, SCIEX BioPharmaView™ software can be used.

As mentioned earlier, organic solvents such as acetonitrile are not suitable for the column and vapor pressure of water is very low. Thus, volatile salts such as 100 mM ammonium acetate or ammonium formate are used. To avoid ion source contamination during prolonged use, molarity should be kept at low (preferably <50 mM). Depending on the need for further optimization for different mAbs, volatile salt ammonium bicarbonate can also be used as such or in combination with other volatile salts.

#### **9. Analysis of Fc fragment on TSKgel FcR-IIIA-NPR**

The C-terminal part of the heavy chains contains the Fc fragment which is responsible for cellular effector functions, essential for proper function of most therapeutic mAbs. In some cases, it is desirable to express fragment antibodies that are smaller than intact mAbs but still are capable of eliciting their therapeutic function by activation of the immune system. Literature reports that both glycoengineering and protein engineering have rendered Fc domains with enhanced Fc receptor binding. In general, Fc fragments and their numerous variants are rapidly gaining interest as a platform in the development of efficient biotherapeutics.

The binding efficiency of the Fc fragment was tested in-house to assess suitability of TSKgel FcR-IIIA-NPR column on the characterization of smaller fragment antibodies. In short, trastuzumab was fragmented with papain that cleaves IgG at His228 forming two Fab parts and one Fc part (**Figure 26**). The reaction mixture was incubated at 37°C for 15 minutes to activate papain followed by mAb addition and further incubation overnight at room temperature. Papain activity was stopped with 5 mM iodoacetamide. A control a sample (no papain during incubation) and a sample from papain digestion were used for this study. Size exclusion chromatography followed by mass spectrometric analysis confirmed >95% cleavage of mAb to Fc and Fab fragments (data available by request).

Both the control mAb and fragments were analyzed on TSKgel FcR-IIIA-NPR column. As expected, Fab did not bind to the FcR column but eluted in

*Monoclonal Antibodies*

yield and ADCC activity.

**Figure 24.**

as antibacterial agent.

**TSKgel FcR-IIIA-NPR column**

**7.4 Column pH stability and cleaning**

fermentation. Thus, by monitoring samples from the bioreactor using TSKgel FcR-IIIA-NPR, process engineers can approximate the optimal day for desired

*Acid stability of a recombinant Fc*γ*RIII ligand as compared to a wild type ligand***.**

Recommended working pH for the FcR column as mentioned in operational conditions and specifications (OCS) is from pH 8 to 4. As mentioned earlier, the protein ligand contains eight amino acid substitutions for improved stability. To further test acid stability, the column was held at pH3 for 200 hours. The modified ligand did not lose its binding affinity and the selectivity while the wild-type lost the binding affinity and selectivity within one hour (**Figure 24**). Based on this and other studies, a pH range of 3–8 is can be used for short term and pH 4.5–7 for long term usage. Due to a protein nature of the ligand, acetonitrile and other organic solvents are not suitable for the column. For cleaning, 3–5 injections of 0.5–2 mL of a buffer containing 500 mM NaCl or 20% ethanol can safely be used in reverse direction at half the normal flow rate. Once the cleaning procedure is complete, it is necessary to equilibrate the column in mobile phase for at least 45 minutes. Cleaning with alkalic solutions above pH 8 are not recommended since this will denature the protein ligand. Sodium azide (0.05%) can be used in the mobile phase

**8. Mass spectrometric characterization of glycoform peaks separated by** 

Mass spectrometric characterization is becoming an integral part of the liquid chromatography analysis. As an example how TSKgel FcR-IIIA-NPR column can be utilized in mass spec work, we describe here an in-line LC–MS intact mAb analysis of trastuzumab (Herceptin Biosimilar). The analysis was carried out using 100 mM volatile ammonium acetate buffer and a linear pH gradient from pH 6.5 to pH 4.5 at the flow rate of 0.4 mL/min. The wavelenght of detection was 280 nm. The column temperature during the analysis was maintained at 20°C. Three glycoform peaks could be detected by UV detector. Mass spectrometric detection was carried out using SCIEX X500B Q-TOF in ESI positive mode, within mass/charge (m/z) range of 5000–7000. Ion source gases 1 and 2 were maintained at 50 psi, curtain gas at 30 psi, CAD gas at 7 psi and temperature at 450°C. Spray voltage was maintained at 5200 V, declustering

**98**

**Figure 26.** *Schematic representation of monoclonal antibody fragmentation with papain.*

**Figure 27.** *Analysis of intact mAb and Fc fragment on TSKgel FcR-IIIA-NPR column.*

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