Prospects for ion chromatography in quality assessment of biologicals

Quantitative characterisation of excipients in biologicals is an important part of the quality assurance process both at the level of finished products and intermediates, as well as active pharmaceutical ingredients. Ion chromatography with amperometric and conductometric detection of separation products has a number of advantages. The main of the advantages is the possibility of direct determination of semivolatile compounds that have neither chromophoric groups, nor intrinsic fluorescence. The aim of this study was to compare ion chromatography with alternative methods in order to identify promising areas for its use in assessing the quality of biologicals. The authors analysed regulatory documents and literature and summarised the methods applied for quantitative determination of ionic excipients in biological medicinal products. The authors investigated the possibility of using ion chromatography for determination of the main active pharmaceutical ingredient in polysaccharide vaccines and excipients in biologicals. The study demonstrated the feasibility of ion chromatography for simultaneous quantitation of cations (ammonium, calcium, magnesium) and anions (chlorides, sulfates, nitrates) in reconstitution solvents for lyophilised biologicals; quality assessment of active pharmaceutical ingredients in biologicals (quantitative analysis of polysaccharides in polysaccharide vaccines, profiling of glycosylated proteins, etc.); and determination of several carbohydrate stabilisers in biologicals with the same analytical procedure. According to the conclusions, ion-exchange chromatography with conductometric and amperometric detection, aimed at quality assessment of biological products, can shortly take a leading position in quantitation of ionic excipients, carbohydrate stabilisers, and main active ingredients (polysaccharides) in polysaccharide vaccines, including the vaccines in the immunisation schedule.

1. Small H, Stevens TS, Bauman WC. Novel ion exchange chromatographic method using conductimetric detection. Anal Chem. 1975;47(11):1801–9. https://doi.org/10.1021/ac60361a017

2. Becker Y. Chromatography. Instrumental analytics. Methods of chromatography and capillary electrophoresis: monograph. Moscow: Technosphere, 2009 (in Russ.)

3. Skelly NE. Separation of inorganic and organic anions on reversed-phase liquid chromatography columns. Anal Chem. 1982;54(4):712–5. https://doi.org/10.1021/ac00241a026

4. Miao S, Xie P, Mao Z, Fan L, Liu X, Zhou Y, et al. Identification of multiple sources of the acidic charge variants in an IgG1 monoclonal antibody. Appl Microbiol Biotechnol. 2017;101(14):5627–38. https://doi.org/10.1007/s00253-017-8301-x

5. Wang G, Tomasella FP. Ion-pairing HPLC methods to determine EDTA and DTPA in small molecule and biological pharmaceutical formulations. J Pharm Analysis. 2016;6(3):150–6. https://doi.org/10.1016/j.jpha.2016.01.002

6. Shibue M, Mant CT, Hodges RS. Effect of anionic ion-pairing reagent hydrophobicity on selectivity of peptide separations by reversed-phase liquid chromatography. J Chromatogr A. 2005;1080(1):68–75. https://doi.org/10.1016/j.chroma.2005.03.035

7. Åsberg D, Langborg Weinmann A, Leek T, Lewis RJ, Klarqvist M, Leśko M, et al. The importance of ion-pairing in peptide purification by reversed-phase liquid chromatography. J Chromatogr A. 2017;1496;80–91. https://doi.org/10.1016/j.chroma.2017.03.041

8. Leblanc Y, Ramon C, Bihoreau N, Chevreux G. Charge variants characterization of a monoclonal antibody by ion exchange chromatography coupled on-line to native mass spectrometry: case study after a long-term storage at +5 °C. J Chromatogr B. 2017;1048:130–9. https://doi.org/10.1016/j.jchromb.2017.02.017

9. Hebbi V, Chattopadhyay S, Rathore AS. High performance liquid chromatography (HPLC) based direct and simultaneous estimation of excipients in biopharmaceutical products. J Chromatogr B. 2019;1117:118–26. https://doi.org/10.1016/j.jchromb.2019.04.022

10. Lodi G, Storti G, Pellegrini LA, Morbidelli M. Ion exclusion chromatography: model development and experimental evaluation. Ind Eng Chem Res. 2017;56(6):1621–32. https://doi.org/10.1021/acs.iecr.6b04475

11. Yan Y, Liu AP, Wang S, Daly TJ, Li N. Ultrasensitive characterization of charge heterogeneity of therapeutic monoclonal antibodies using strong cation exchange chromatography coupled to native mass spectrometry. Anal Chem. 2018;90(21):13013–20. https://doi.org/10.1021/acs.analchem.8b03773

12. Muneeruddin K, Bobst CE, Frenkel R, Houde D, Turyan I, Sosic Z, Kaltashov IA. Characterization of a PEGylated protein therapeutic by ion exchange chromatography with on-line detection by native ESI MS and MS/MS. Analyst. 2017;142(2):336–44. https://doi.org/10.1039/C6AN02041K

13. Fekete S, Beck A, Fekete J, Guillarme D. Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part I: salt gradient approach. J Pharm Biomed Anal. 2015;102:33–44. https://doi.org/10.1016/j.jpba.2014.08.035

14. Fekete S, Beck A, Fekete J, Guillarme D. Method development for the separation of monoclonal antibody charge variants in cation exchange chromatography, Part II: pH gradient approach. J Pharm Biomed Anal. 2015;102:282–9. https://doi.org/10.1016/j.jpba.2014.09.032

15. Spanov B, Olaleye O, Lingg N, Bentlage AEH, Govorukhina N, Hermans J, et al. Change of charge variant composition of trastuzumab upon stressing at physiological conditions. J Chromatogr A. 2021;1655:462506. https://doi.org/10.1016/j.chroma.2021.462506

16. Vlasak J, Bussat MC, Wang S, Wagner-Rousset E, Schaefer M, Klinguer-Hamour C, et al. Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody. Anal Biochem. 2009;392(2):145–54. https://doi.org/10.1016/j.ab.2009.05.043

17. Faghihi H, Merrikhihaghi S, Najafabadi AR, Ramezani V, Sardari S, Vatanara A. A comparative study to evaluate the effect of different carbohydrates on the stability of Immunoglobulin G during lyophilization and following storage. Pharm Sci. 2016;22(4):251–9. https://doi.org/10.15171/PS.2016.39

18. Wlodarczyk SR, Custódio D, Pessoa A Jr, Monteiro G. Influence and effect of osmolytes in biopharmaceutical formulations. Eur J Pharm Biopharm. 2018;131:92–8. https://doi.org/10.1016/j.ejpb.2018.07.019

19. Kissinger PT, Refshauge C, Dreiling R, Adams RN. An electrochemical detector for liquid chromatography with picogram sensitivity. Anal Lett. 1973;6(5):465–77. https://doi.org/10.1080/00032717308058694

20. Merkle RK, Poppe I. Carbohydrate composition analysis of glycoconjugates by gas-liquid chromatography/mass spectrometry. Methods Enzymol. 1994;230:1–15. https://doi.org/10.1016/0076-6879(94)30003-8

21. Schenk J, Nagy G, Pohl NLB, Leghissa A, Smuts J, Schug KA. Identification and deconvolution of carbohydrates with gas chromatography-vacuum ultraviolet spectroscopy. J Chromatogr A. 2017;1513:210–21. https://doi.org/10.1016/j.chroma.2017.07.052

22. Haas M, Lamour S, Trapp O. Development of an advanced derivatization protocol for the unambiguous identification of monosaccharides in complex mixtures by gas and liquid chromatography. J Chromatogr A. 2018;1568:160–7. https://doi.org/10.1016/j.chroma.2018.07.015

23. Rendleman JA, Jr. In: Isbell HS, ed. Carbohydrates in solution. Advances in Chemistry Ser ACS. Washington; 1973;117:51–68.

24. Hardy MR, Townsend RR, Lee YC. Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection. Anal Biochem. 1988;170(1):54–62. https://doi.org/10.1016/0003-2697(88)90089-9

25. Rohrer JS, Basumallick L, Hurum DC. Profiling N-linked oligosaccharides from IgG by high-performance anion-exchange chromatography with pulsed amperometric detection. Glycobiology. 2016;26(6):582–91. https://doi.org/10.1093/glycob/cww006

26. Talaga P, Vialle S, Moreau M. Development of high-performance anion-exchange chromatography with pulsed-amperometric detection based quantification assay for pneumococcal polysaccharides and conjugates. Vaccine. 2002;20(19-20):2474–84. https://doi.org/10.1016/S0264-410X(02)00183-4

27. Gudlavalleti SK, Crawford EN, Harder JD, Reddy JR. Quantification of each serogroup polysaccharide of Neisseria meningitidis in A/C/Y/W-135-DT conjugate vaccine by high-performance anion-exchange chromatography-pulsed amperometric detection analysis. Anal Chem. 2014;86(11):5383−90. https://doi.org/10.1021/ac5003933

28. van der Put RM, de Haan A, van den IJssel JG, Hamidi A, Beurret M. HPAEC-PAD quantification of Haemophilus influenzae type b polysaccharide in upstream and downstream samples. Vaccine, 2015;33(48):6908–13. https://doi.org/10.1016/j.vaccine.2014.07.028