Best Practices for Maintaining Column Performance in Size-Exclusion Chromatography during Long-Term Storage

Significance of the Topic

Long-term storage of size-exclusion chromatography (SEC) columns is critical for preserving their resolving power, especially when analyzing sensitive protein therapeutics. Improper storage solvent composition can lead to microbial contamination, changes in retention times, loss of high molecular weight species (HMWS), and reduced resolution of low molecular weight species (LMWS). Ensuring reliable column performance over months of inactivity supports robust quality control in biopharmaceutical research and manufacturing.

Objectives and Study Overview

This study evaluated five candidate storage solvents for Waters BioResolve SEC mAb Columns by monitoring column performance at 1, 2, and 4 months at ambient temperature. Performance metrics focused on percent HMWS recovery, resolution between dimer and monomer (USP Res. HH), and LMWS quantitation and resolution. A direct comparison to the previously recommended 20% methanol/80% water solvent was included to support the selection of an optimized shipping and storage solution.

Methodology and Instrumentation

Fifteen BioResolve SEC mAb columns (200 Å, 2.5 µm, 4.6×150 mm) were initially characterized using an ACQUITY UPLC H-Class Bio system with Tunable UV detection at 280 nm. The Waters mAb Size Variant Standard, containing NISTmAb RM 8671 and IdeS fragments, was injected under controlled temperature and flow conditions. Columns were flushed with 10 CV of one of five storage solvents:

• A: 10% ACN/90% 25 mM Na-PO4 pH 7 + 100 mM KCl
• B: 10% ACN/90% 2.5 mM Na-PO4 pH 7 + 10 mM KCl
• C: 10% ACN/90% 20 mM Na-PO4 pH 6.8 (no salt)
• D: 10% ACN/90% water
• E: 20% methanol/80% water

After storage, one column per group was retested at each time point. Additional corrosion assessments of 316 SS frits were performed by extended soaking and microscopic inspection up to nine months.

Main Findings and Discussion

All solvents preserved baseline performance for one month. By two to four months, buffer- and salt-free solvents (C and D) showed progressive decline in HMWS recovery (up to 66% loss), reduced dimer-monomer resolution (up to 44% decrease in USP Res. HH), and degraded LMWS resolution. The standard methanol solvent (E) exhibited similar but less severe deterioration. In contrast, solvents A and B maintained HMWS and LMWS metrics within acceptable limits over four months. Microscopic examination and accelerated corrosion tests confirmed that 10% ACN with phosphate buffer and KCl is non-corrosive to austenitic 316 SS frits.

Benefits and Practical Applications

• Validated a non-toxic bacteriostatic storage solvent (10% ACN in phosphate buffer with KCl) that ensures consistent SEC column performance.
• Confirmed compatibility with stainless-steel components, alleviating concerns about chloride-induced corrosion.
• Provided evidence to replace methanol-based storage with an optimized ACN-buffer-salt mixture for biopharmaceutical QC workflows.

Future Trends and Opportunities

Emerging research may explore broadening buffer and salt combinations to fine-tune antimicrobial efficacy and column stability. Alternative organic co-solvents with enhanced bactericidal properties could further reduce storage risk. Integration of real-time microbial sensors in storage vials and automated column conditioning protocols may streamline long-term maintenance. Advances in column materials and surface coatings may also mitigate fouling and extend column lifetimes.

Conclusion

The study demonstrates that long-term storage solvents containing both buffer and salt in a 10% acetonitrile background best preserve SEC column performance for mAb analysis. The recommended formulation—10% ACN, 25 mM sodium phosphate pH 7, 100 mM KCl—balances antimicrobial protection with non-corrosive behavior, ensuring reliable monitoring of protein aggregates and fragments after extended inactivity.

References

1. Hotchkiss M. Studies on Salt Action: VI. The Stimulating and Inhibitive Effect of Certain Cations upon Bacterial Growth. J Bacteriol. 1923;8(2):141–162.
2. Fabian FW, Winslow CE. The Influence upon Bacterial Viability of Various Anions in Combination with Sodium. J Bacteriol. 1929;18(4):265–291.
3. Holm GE, Sherman JM. Salt Effects in Bacterial Growth: I. Preliminary Paper. J Bacteriol. 1921;6(6):511–519.
4. Lichstein HC, Soule MH. Studies of the Effect of Sodium Azide on Microbic Growth and Respiration: I. The Action of Sodium Azide on Microbic Growth. J Bacteriol. 1944;47(3):221–230.
5. Maguire ED, Wallis RB. The Role of Bacterial Contamination in the Isolation of Apparent Anti-Inflammatory Factors from Rabbit Anti Lymphocytic Serum. Br J Pharmacol. 1977;59:261–268.
6. Kongpol A, Kato J, Tajima T, Vangnai AS. Characterization of Acetonitrile-Tolerant Marine Bacterium Exiguobacterium sp. Microbes Environ. 2012;27(1):30–35.
7. Xu G, Wu A, Xiao L, Han R, Ni Y. Enhancing Butanol Tolerance of Escherichia coli Reveals Hydrophobic Interaction of Multi-Tasking Chaperone SecB. Biotechnol Biofuels. 2019;12:164.
8. NIOSH. Registry of Toxic Effects of Chemical Substances (RTECS): Sodium Azide. VY8050000.
9. Bräse S, Mende M, Jobelius HH, Scharf H-D. Hydrazoic Acid and Azides. Ullmann’s Encyclopedia of Industrial Chemistry. 2015.
10. Chang S, Lamm SH. Human Health Effects of Sodium Azide Exposure: A Literature Review and Analysis. Int J Toxicol. 2003;22(3):175–186.
11. NIOSH. Current Intelligence Bulletin 13: Explosive Azide Hazard. 1976.
12. Millipore Corp. Guide to Successful Operation of Your LC System: Mobile Phase Preparation and Use. Manual 022378 Rev 0. 1991.
13. Meyer VR. Practical High-Performance Liquid Chromatography. 3rd ed. John Wiley & Sons; 1998.
14. Link GW Jr, Keller PL. Effects of Solutions Used for Storage of Size-Exclusion Columns on Subsequent Chromatography of Peptides and Proteins. J Chromatogr. 1985;331:253–264.