8. Size exclusion chromatography column in the HILIC mode

For many years, SEC columns have been used to separate various nucleic acid species such as DNA, RNA, and tRNA as well as their constituent bases, adenine, guanine, thymine, cytosine, and uracil. In medicine, several primary nucleobases are the basis for the nucleoside analogues and other synthetic analogs which are used as anticancer and antiviral agents. Nucleobase modifications are the basis of oligonucleotide-based therapeutics, making their purification very important.

Hydrophilic interaction chromatography (HILIC) is a variant of normal phase liquid chromatography which uses hydrophilic stationary phases with reversed-phase type eluents. It is applied for the separation of polar hydrophilic compounds.

Chemically bonded diol-coated phases in size exclusion chromatography (SEC) columns demonstrate high polarity and hydrogen bonding properties. They do not contain ionizable groups compared to the unreacted free residual silanols, making them appropriate for the HILIC mode.

Figure 34. Separation of four nucleobases using a TSKgel SuperSW mAb HTP column under SEC condition (left panel) and in HILIC mode (right panel) at pH 7.4.

mentioned that sodium azide (NaN3) was used as an antibacterial agent to prevent fouling of

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During the SEC of mAbs and proteins, mobile phases containing salts are constantly used and can deposit on the different parts of the HPLC system (Figure 36). The pump head assembly may get affected from the deposition of salts, resulting into a rise of back pressure as well as causing damage to the sapphire piston. The pump head assembly can be cleaned with distilled water and the back pressure issue is resolved. Mobile phase containing high salt or a combination of high salt and organic solvent can create this problem more than any other low salt aqueous mobile phases. The system should not remain idle with these types of mobile phases. The deposition may happen anywhere within the HPLC system and also inside the column, or

The use of a surfactant is necessary for certain SEC applications as discussed earlier, but surfactants may change the bonding phase, so it may be necessary to dedicate the column for

Occasionally protein samples are adsorbed onto the packing material. When this occurs, it is time to clean the SEC column. At the beginning of the separation, when the SEC column is new and operating correctly and the mAb monomer is yielding a well-resolved peak, it is better to establish baseline data and acceptable running conditions criteria. Then, if one of the performance

the phosphate buffer.

Figure 35. Comparison of a dirty frit with a new frit.

that particular application.

Figure 36. Salt depositions at screws of the column.

in the pump, the injector, the in-line filter or the tubing.

Figure 34 [44] shows the separation of four nucleobases using a TSKgel SuperSW mAb HTP Column under SEC condition and in HILIC mode at pH 7.4 (Figure 34).

As expected, due to the similarities in molecular masses between the four compounds, significant interference is observed among the peaks of interest, particularly the three pyrimidine derivatives, when separated on the TSKgel SuperSW mAb HTP column under SEC conditions. The late elution of adenine (relative to the other three compounds) may be attributed to possible interactions between the stationary phase and the derivatized purine compound, leading to a shift toward a longer retention time. When the same SEC column is used in the HILIC mode, the order of elution of the analytes does not correlate with their molecular mass (as in SEC separations), but instead is based on their relative hydrophilicity. This note demonstrates the benefits of using a SEC column in HILIC mode for the superior resolution of four nucleobases, as opposed to using the column in the SEC mode or using another type of a HILIC column.

#### 9. Tips and tricks for size exclusion chromatography

It is a recommended practice to protect the column from potential sources of contamination during the SEC separation of mAbs and other proteins. Standards and mobile phases should be filtered through a 0.45-μm syringe filter. A frit filter used between injector and column will also be an additional help. The use of guard columns is highly recommended. Guard columns being short, of similar ID and with the same stationary phase do not possess any separation power. The slight change in the retention time due to small increase in length remains constant in consecutive injections. Using guard columns can prolong the lifetime of the analytical column. The guard column needs to be changed before the dirty material spills over to the analytical column. Frits at different parts of the HPLC instruments need to be changed intermittently. Please refer to the picture below which clearly shows how much dirty materials are trapped by these frits (Figure 35). Frequent changes of the frits are necessary to avoid spillage of the dirty materials to the columns. Phosphate buffer pH 6.8, very commonly used for the protein analysis by SEC is prone to bacterial growth. The column will get clogged and dirty, eventually leading to failure of the analysis and breakdown of the system. Previously I

Figure 35. Comparison of a dirty frit with a new frit.

Figure 34 [44] shows the separation of four nucleobases using a TSKgel SuperSW mAb HTP

Figure 34. Separation of four nucleobases using a TSKgel SuperSW mAb HTP column under SEC condition (left panel)

As expected, due to the similarities in molecular masses between the four compounds, significant interference is observed among the peaks of interest, particularly the three pyrimidine derivatives, when separated on the TSKgel SuperSW mAb HTP column under SEC conditions. The late elution of adenine (relative to the other three compounds) may be attributed to possible interactions between the stationary phase and the derivatized purine compound, leading to a shift toward a longer retention time. When the same SEC column is used in the HILIC mode, the order of elution of the analytes does not correlate with their molecular mass (as in SEC separations), but instead is based on their relative hydrophilicity. This note demonstrates the benefits of using a SEC column in HILIC mode for the superior resolution of four nucleobases, as opposed

It is a recommended practice to protect the column from potential sources of contamination during the SEC separation of mAbs and other proteins. Standards and mobile phases should be filtered through a 0.45-μm syringe filter. A frit filter used between injector and column will also be an additional help. The use of guard columns is highly recommended. Guard columns being short, of similar ID and with the same stationary phase do not possess any separation power. The slight change in the retention time due to small increase in length remains constant in consecutive injections. Using guard columns can prolong the lifetime of the analytical column. The guard column needs to be changed before the dirty material spills over to the analytical column. Frits at different parts of the HPLC instruments need to be changed intermittently. Please refer to the picture below which clearly shows how much dirty materials are trapped by these frits (Figure 35). Frequent changes of the frits are necessary to avoid spillage of the dirty materials to the columns. Phosphate buffer pH 6.8, very commonly used for the protein analysis by SEC is prone to bacterial growth. The column will get clogged and dirty, eventually leading to failure of the analysis and breakdown of the system. Previously I

Column under SEC condition and in HILIC mode at pH 7.4 (Figure 34).

and in HILIC mode (right panel) at pH 7.4.

168 Antibody Engineering

to using the column in the SEC mode or using another type of a HILIC column.

9. Tips and tricks for size exclusion chromatography

mentioned that sodium azide (NaN3) was used as an antibacterial agent to prevent fouling of the phosphate buffer.

During the SEC of mAbs and proteins, mobile phases containing salts are constantly used and can deposit on the different parts of the HPLC system (Figure 36). The pump head assembly may get affected from the deposition of salts, resulting into a rise of back pressure as well as causing damage to the sapphire piston. The pump head assembly can be cleaned with distilled water and the back pressure issue is resolved. Mobile phase containing high salt or a combination of high salt and organic solvent can create this problem more than any other low salt aqueous mobile phases. The system should not remain idle with these types of mobile phases. The deposition may happen anywhere within the HPLC system and also inside the column, or in the pump, the injector, the in-line filter or the tubing.

The use of a surfactant is necessary for certain SEC applications as discussed earlier, but surfactants may change the bonding phase, so it may be necessary to dedicate the column for that particular application.

Occasionally protein samples are adsorbed onto the packing material. When this occurs, it is time to clean the SEC column. At the beginning of the separation, when the SEC column is new and operating correctly and the mAb monomer is yielding a well-resolved peak, it is better to establish baseline data and acceptable running conditions criteria. Then, if one of the performance

Figure 36. Salt depositions at screws of the column.

characteristics of the SEC column changes by 10% or more, it is prudent that cleaning is necessary. The acceptance criteria can be more stringent as needed. Similar cleaning of the whole system including the flow cell may be necessary.

stationary phase as well as by optimizing the mobile phase. The effect of particle size and pore size of the stationary phase on SEC separation needs to be clearly understood. A right column selection for the purification will depend on the separation range and the slope of the calibration curve. Separation of HMW and LMW species to purify the monomer is critical to get the pure mAb without heterogenic impurities, which might be immunogenic to human. Enzymatic digestion and forced degradations are necessary to characterize the monoclonal antibodies, and SEC columns need to be rightfully selected based on the separation goal. Mobile phase additives may be necessary to improve the separation, and few examples are discussed. Since online LC/MS is becoming popular, a mobile phase compatibility is needed as discussed in this chapter. Since size exclusion chromatography cannot separate the heterogenic species without much difference in hydrodynamic radii, an orthogonal technique is necessary. A similarly complimentary technique is also helpful for complete characterization of the monoclonal antibodies. SEC column developed, and optimization of surface and pore characteristics is critical for the separation of monoclonal antibodies and other proteins by HPLC and UHPLC instrument. With the advancement of UHPLC, an easy method transfer between HPLC and UHPLC is becoming necessary. Ease of method transfer using a column with dual functionality for its use in both HPLC and UHPLC is helpful. Desalting using a conventional SEC HPLC column can be very useful in removing the unwanted salts and additives. A robust separation with excellent reproducibility needs a size exclusion chromatography column with optimized packing as well as the knowledge about the tips and tricks to maintain the column lifetime.

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Yoshimi Hashimoto, Toshiaki Nishi, Kazuaki Muranaka, Kosuke Araki, Yasutami Mitoma, Shigeru Nakatani, Hasegawa, Sachiyuki, Oscar Yamasaki, Toru Satoh – Tosoh Corporation, Separation Centre, Japan for their contributions in a number of references and overall support. Tosoh Bioscience LLC and Tosoh Corporation, Tosoh Bioscience LLC, Tosoh Bioscience Gmbh.

[1] Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The Immune System in

Acknowledgements

Author details

Atis Chakrabarti

References

Tosoh Bioscience LLC, USA

Address all correspondence to: atis.chakrabarti@tosoh.com

Health and Disease. 5th ed. New York: Garland Science; 2001

If cleaning is necessary, it is better to try cleaning the column in reversed direction at half the flow rate while the column is detached from the detector to prevent detector damage since the proteins have a tendency to adhere to the quartz material of the detector. Since the dirty materials may remain trapped over the frit outside the column, cleaning in reversed flow direction may be the easiest way to clean the column. Once the column is cleaned using the vendor suggested protocol, the analyst must qualify the column using the QC method and QC pass criteria under normal flow direction.

It is always recommended to read the operational conditions and specification sheet (OCS) before the column is used. Occasionally, we may intend to use the column outside the specification for a short period of time, but it is the responsibility of the analyst to monitor if the column is functioning properly. For example, in Section 2.5, it has been shown that an analysis could be repeated to monitor the stability of the antibody over time using a TSKgel SuperSW mAb-HTP column at 0.75 mL/min. The used flow rate (0.75 mL/min) was higher than the recommended maximum flow rate of 0.5 mL/min, as mentioned in the operational conditions and specification (OCS) sheet for this column. Though the column could successfully be used at this higher flow rate without compromising the column health, the effect of the higher flow rate on the column lifetime for prolonged use was not investigated. It is always wise to operate within recommended maximum flow rate to remain in safe side in order to prolong the lifetime of the column. It is always necessary to keep an eye on the back pressure, peak parameters, and so on to monitor the column health.

HPLC system and analytical chromatography columns are costly. Method development is time-consuming and costly too. It is important to employ an HPLC system that is optimized with regard to extra-column band broadening to take full advantage of the high column efficiency that can be obtained on analytical columns. For any troubleshooting situation, the problem is one of the following: [1] the column, [2] the sample, [3] the mobile phases, [4] the instrumentation, and [5] personal errors. The same is true for size exclusion chromatography related troubleshooting issues too. Proper maintenance of the column and instrument is much more needed and important to avoid the troubleshooting in the beginning. Many troubleshooting issues can be avoided by taking proper handling the top three major components of any HPLC analysis. The importance of the use of a protein standard mixture and the standard mAb to monitor the column health from time to time is always a good idea as mentioned in brief in Section 2.5. Maintaining a SEC column is an art and good performance, which will result in a prolonged column lifetime.

### 10. Conclusions

Size-exclusion chromatography is a great tool for the purification of monoclonal antibodies. The secondary interaction of the stationary phase needs to be taken care of by controlling the stationary phase as well as by optimizing the mobile phase. The effect of particle size and pore size of the stationary phase on SEC separation needs to be clearly understood. A right column selection for the purification will depend on the separation range and the slope of the calibration curve. Separation of HMW and LMW species to purify the monomer is critical to get the pure mAb without heterogenic impurities, which might be immunogenic to human. Enzymatic digestion and forced degradations are necessary to characterize the monoclonal antibodies, and SEC columns need to be rightfully selected based on the separation goal. Mobile phase additives may be necessary to improve the separation, and few examples are discussed. Since online LC/MS is becoming popular, a mobile phase compatibility is needed as discussed in this chapter. Since size exclusion chromatography cannot separate the heterogenic species without much difference in hydrodynamic radii, an orthogonal technique is necessary. A similarly complimentary technique is also helpful for complete characterization of the monoclonal antibodies. SEC column developed, and optimization of surface and pore characteristics is critical for the separation of monoclonal antibodies and other proteins by HPLC and UHPLC instrument. With the advancement of UHPLC, an easy method transfer between HPLC and UHPLC is becoming necessary. Ease of method transfer using a column with dual functionality for its use in both HPLC and UHPLC is helpful. Desalting using a conventional SEC HPLC column can be very useful in removing the unwanted salts and additives. A robust separation with excellent reproducibility needs a size exclusion chromatography column with optimized packing as well as the knowledge about the tips and tricks to maintain the column lifetime.
