Comparative evaluation of purification strategies for anti-rabies immunoglobulins: Impact on yield, functional activity, and stability: Purification of anti-rabies immunoglobulins

Anara Nurmukhambetova; Gulbaram Ustenova; Baikara Barshagul; Assima Abekova; Meruert Zhylkyshybaeva

doi:10.62310/liab.v6i2.339

Authors

Anara Nurmukhambetova Laboratory of Cellular Biotechnology, LLP Research and Production Enterprise “Antigen”, 4 Azerbayev Street, Abay 040905, Karasai District, Almaty Region, Republic of Kazakhstan https://orcid.org/0009-0004-1840-7826
Gulbaram Ustenova Department of Pharmaceutical Technology, Kazakh National Medical University named after S.D. Asfendiyarov, 94 Tole bi Street, Almaty 050012, Republic of Kazakhstan https://orcid.org/0009-0007-6595-5640
Baikara Barshagul LLP Research and Production Enterprise “Antigen”, 4 Azerbayev Street, Abay 040905, Karasai District, Almaty Region, Republic of Kazakhstan https://orcid.org/0009-0003-2921-3202
Assima Abekova LLP Research and Production Enterprise “Antigen”, 4 Azerbayev Street, Abay 040905, Karasai District, Almaty Region, Republic of Kazakhstan https://orcid.org/0009-0003-9040-9247
Meruert Zhylkyshybaeva Department of Animal Biology, Kazakh National Agrarian Research University, 8 Abay Avenue, Almaty 050010, Republic of Kazakhstan https://orcid.org/0009-0000-0434-5872

DOI:

https://doi.org/10.62310/liab.v6i2.339

Keywords:

Anti-rabies immunoglobulins, Purification methods, Biological activity, Protein yield, Pharmaceutical stability, Virus neutralisation

Abstract

The study aimed to evaluate the effectiveness of different purification methods for anti-rabies immunoglobulins obtained from the plasma of hyperimmunised cattle. The study included a total of 30 animals, randomly divided into three groups based on the purification method: ammonium sulphate precipitation, affinity chromatography on a Protein G column, and a combined method with the addition of ultrafiltration. The purification methods differed in their ability to preserve protein yield, specific antibody activity, and virus-neutralising capacity. The combined protocol produced the highest final protein concentration; however, total protein was interpreted only as an indicator of recovery and concentration efficiency, not as evidence of IgG purity. The superiority of the combined method was supported by the parallel increase in anti-rabies IgG reactivity and virus-neutralising activity. In contrast, the method of precipitation with ammonium sulphate revealed the lowest values for each of these parameters, indicating the lower selectivity of this method and the loss of the functionally active immunoglobulins. However, the results obtained with the precipitation method, Protein G affinity chromatography and ultrafiltration suggest that these methods increase both the amount of anti-rabies immunoglobulin that is obtained and the quality of that immunoglobulin. The method of precipitation with ammonium sulphate and the storage of the obtained immunoglobulin without the step of freezing revealed the greatest decrease in each of the indicators over the 60-day period. However, the method that employed the use of all three steps revealed the best results in the preservation of the protein, specific antibodies, and virus- neutralising activity, with only a 4% loss of each of these initial values after storage at -20°C. Statistical analysis using repeated measures ANOVA confirmed the significant influence of the purification method and storage conditions on all the parameters studied (p < 0.05). In conclusion, the integration of precipitation, affinity chromatography, and ultrafiltration resulted in a better yield, functional activity, and stability of anti-rabies immunoglobulins. The practical significance of the present findings lies in the possibility of applying them in the development, standardisation, and production of highly effective immunoglobulin preparations for the prevention and treatment of rabies in veterinary and medical practice.

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References

Bartaquini RT, Torquato RB, Fernandes ER, Guedes F, De Castro BS, Katz ISS, Scheffer KC, De Cassia Rodrigues Da Silva A, Pimenta DC, Silva SR. (2020). Evaluation of polyclonal anti-RNP IgG antibody for rabies diagnosis by indirect rapid immunohistochemistry test. Acta Tropica 206: 105340. https://doi.org/10.1016/j.actatropica.2020.105340

Bitanova E, Akhmetsadykov N, Abdraimova Z, Batanova Z, Baikara B, Bayandy G, Krykbayev Y, Nurmuhambetova A, Imanbekova T. (2024). Methods of anti-rabic immunoglobulin production. Pharmaceutics of Kazakhstan 4: 287-297. https://doi.org/10.53511/pharmkaz.2024.46.70.034

Cai M, Hu Z, Yang Y, Mao T, Liu Y, Lu G, Yang F, Qi J, Huang W, Wang Y. (2023). Characterization of a human-mouse chimeric monoclonal antibody targeting rabies virus glycoprotein. Journal of Medical Virology 95(7): e28954. https://doi.org/10.1002/jmv.28954

Chorpunkul A, Boonyuen U, Limkittikul K, Saengseesom W, Phongphaew W, Putchong I, Chankeeree P, Theerawatanasirikul S, Hajitou A, Benjathummarak S, Pitaksajjakul P, Lekcharoensuk P, Ramasoota P. (2024). Development of novel canine phage display-derived neutralizing monoclonal antibody fragments against rabies virus from immunized dogs. Scientific Reports 14(1): 22939. https://doi.org/10.1038/s41598-024-73339-2

Council of Europe. (1986). European convention for the protection of vertebrate animals used for experimental and other scientific purposes. European Treaty Series – No. 123, Strasbourg, France. https://rm.coe.int/168007a67b

De Melo GD, Hellert J, Gupta R, Corti D, Bourhy H. (2022). Monoclonal antibodies against rabies: Current uses in prophylaxis and in therapy. Current Opinion in Virology 53: 101204. https://doi.org/10.1016/j.coviro.2022.101204

Ejemel M, Smith TG, Greenberg L, Carson WC, Lowe D, Yang Y, Jackson FR, Morgan CN, Martin BE, Kling C, Hutson CL, Gallardo-Romero N, Ellison JA, Moore S, Buzby A, Sullivan-Bolyai J, Klempner M, Wang Y. (2022). A cocktail of human monoclonal antibodies broadly neutralizes North American rabies virus variants as a promising candidate for rabies post-exposure prophylaxis. Scientific Reports 12(1): 9403. https://doi.org/10.1038/s41598-022-13527-0

Fan L, Zhang L, Li J, Zhu F. (2022). Advances in the progress of monoclonal antibodies for rabies. Human Vaccines & Immunotherapeutics 18(1): 2026713. https://doi.org/10.1080/21645515.2022.2026713

Fontana D, Rodriguez MC, Garay E, Russo S, Prieto C. (2020). Optimization and validation of a blocking ELISA for quantitation of anti-rabies immunoglobulins in multispecies sera. Applied Microbiology and Biotechnology 104(9): 4127-4139. https://doi.org/10.1007/s00253-020-10490-6

Ginayatov N, Aitpayeva Z, Zhubantayev I, Kassymbekova L, Zhanabayev A, Abulgazimova G, Arynova R, Bizhanov A, Mussayeva A, Berdikulov M, Aisin M, Sayakova Z, Tursunkulov S, Rametov N, Akhmadiyeva A, Bulasheva A, Jussupbekova N, Yeskhojayev O, Baikadamova G, Kushaliyev K, Burambayeva N, Issimov A. (2025). Smallholder cattle farmers’ knowledge, attitudes, and practices toward rabies: A regional survey in Kazakhstan. Veterinary Sciences 12(4): 335. https://doi.org/10.3390/vetsci12040335

Hou Q, Liu S, Liu C, Wang X, Shi J, Chen Q, Lv X, Zhu Z, Wang C, Yin W. (2024). Immunogenicity of 4-dose Essen intramuscular regimen for rabies post-exposure prophylaxis: A multi-center cross-sectional study in China. Travel Medicine and Infectious Disease 60: 102735. https://doi.org/10.1016/j.tmaid.2024.102735

Kansagra K, Parmar D, Mendiratta SK, Patel J, Joshi S, Sharma N, Parihar A, Bhoge S, Patel H, Kalita P, Munshi R, Kurmi P, Shah R, Gupta A, Bhalla H, Bekkalele H, Verma R, Agarwal D, Sharma S, Gawande A, Chhaya G. (2020). A phase 3, randomized, open-label, noninferiority trial evaluating anti-rabies monoclonal antibody cocktail (TwinRABTM) against human Rabies immunoglobulin (HRIG). Clinical Infectious Diseases 73(9): e2722-e2728. https://doi.org/10.1093/cid/ciaa779

Liu X, Li Y, Li J, Zhou J, Guo J, Pu Y, Jiang Y, Zhou Y, Jiang Z, Shu Q, Wang C, Wang J, Zhao Y, Zhao W, Wang H, Wei J, Yu H, Gao J, Li X. (2023). Comparing recombinant human rabies monoclonal antibody (ormutivimab) with human rabies immunoglobulin (HRIG) for postexposure prophylaxis: A phase III, randomized, double-blind, non-inferiority trial. International Journal of Infectious Diseases 134: 53-62. https://doi.org/10.1016/j.ijid.2023.05.017

Lumlertdacha B, Mahong B, Rattanapisit K, Bulaon CJI, Hemachudha T, Phoolcharoen W. (2023). Efficiency comparative approach of plant-produced monoclonal antibodies against rabies virus infection. Vaccines 11(8): 1377. https://doi.org/10.3390/vaccines11081377

Lupitha SS, Varghese GR, Das LJ, Prabhakaran P, Ananda AM, Mani RS, Sreekumar E. (2025). Assessing anti-rabies vaccine response in humans: A rapid and high-throughput adaptable, pseudovirus-based neutralization assay as an alternative to rapid fluorescence focus inhibition test (RFFIT). PLoS Neglected Tropical Diseases 19(4): e0013010. https://doi.org/10.1371/journal.pntd.0013010

Manalo DL, Gomez MR, Jarilla BR, Ang MJC, Tuason LT, Demetria CS, Medina PB, Dilig JE, Avenido-Cervantes EF, Park C, Inoue S. (2020). A preliminary evaluation of a locally produced biotinylated polyclonal anti-rabies antibody for direct rapid immunohistochemical test (DRIT) in the Philippines. Acta Tropica 211: 105610. https://doi.org/10.1016/j.actatropica.2020.105610

Mo H, Chen Q, Zhang Z, Lin G, Wang Y, Mo L, Liang W, He J, Su B, Li J, Hu Z, Li H, Pei N, Wu Y, Su H. (2025). Development of a blocking ELISA for evaluating neutralizing antibodies in human and canine serum based on rabies virus glycoprotein epitope I. International Journal of Biological Macromolecules 301: 140275. https://doi.org/10.1016/j.ijbiomac.2025.140275

Nurmukhambetova A, Batanova Z, Bayandy G, Krykbayev Y, Bitanova E, Akhmetsadykov N, Amankeldi D. (2025). Modes for chromatographic purification of immunoglobulin G (IgG) from the serum of horses hyperimmunized with rabies antigen. Fundamental and Experimental Biology 30(1): 36-43. https://doi.org/10.31489/2025bmg1/36-43

Pati I, Cruciani M, Candura F, Massari MS, Piccinini V, Masiello F, Profili S, De Fulvio L, Pupella S, De Angelis V. (2023). Hyperimmune globulins for the management of infectious diseases. Viruses 15(7): 1543. https://doi.org/10.3390/v15071543

Quiambao BP, Payumo RA, Roa C, Borja-Tabora CF, Montellano ME, De Los Reyes MR, Jesus LZ, Capeding MR, Solimen DP, Barez MY, Reid C, Chuang A, Tsao E, McClain JB. (2024). A phase 2b, randomized, double blinded comparison of the safety and efficacy of the monoclonal antibody mixture SYN023 and human rabies immune globulin in patients exposed to rabies. Vaccine 42(22): 126018. https://doi.org/10.1016/j.vaccine.2024.05.066

Rathnadiwakara H, Amarasekara S, Cliquet F, Thibault J, Jalaldeen RN, Gunatilake M. (2025). Cellular immune response against rabies vaccination in laboratory-bred dogs: A systematised review. Laboratory Animals 59(3): 342-355. https://doi.org/10.1177/00236772241300782

Santosh AK, Kumar D, Kaur C, Gupta P, Jasmeen P, Dilip L, Kavitha G, Basagoudanavar S, Hosamani M, Balamurugan V, Sharada R, Rathnamma D, Sunil KM, Hegde NR, Isloor S. (2024). Evaluation of the immune status of dogs vaccinated against rabies by an enzyme-linked immunosorbent assay using crude preparations of insect cells infected with a recombinant baculovirus encoding the rabies virus glycoprotein gene. PLoS One 19(12): e0314516. https://doi.org/10.1371/journal.pone.0314516

Soliman R, Hashem Z, El-Hariri M, Elyazeed HA, Aboul-Ella H. (2022). Production of Rabies virus-specific monoclonal antibodies and evaluation of their neutralizing potential. bioRxiv (Cold Spring Harbor Laboratory). https://doi.org/10.1101/2022.11.24.517809

Woznichak M, Vandeberg P, Russ C, Talton C, Srivastava J, Arora V, Merritt WK, Jose M. (2021). Application of a caprylate/chromatography purification process for production of a high potency rabies immune globulin from pooled human plasma. Journal of Immunological Methods 499: 113164. https://doi.org/10.1016/j.jim.2021.113164

Wu L, Zhang Y, Huang Z, Lu H, Li X, Zhu Q, Yin C, Liu J, Shao H, Sun X. (2025). Antibody persistence of human diploid cell rabies vaccine administrated using the four-versus five-dose ESSEN intramuscular regimen in post-exposure prophylaxis: A prospective cohort study among the Chinese population. Vaccines 13(3): 215. https://doi.org/10.3390/vaccines13030215

Wu M, Peng X, Xu W, Zhang H, Fang L, Liu X, Miao F, Liu Q, Mi S, Xiao Y, Yu X, Tu C, Ge L, Liu Y. (2024). Development of human monoclonal antibodies with broad reactivity for rabies postexposure prophylaxis. Journal of Medical Virology 96(11): e70068. https://doi.org/10.1002/jmv.70068

Yumoto K, Arisaka T, Okada K, Aoki K, Ose T, Masatani T, Sugiyama M, Ito N, Fukuhara H, Maenaka K. (2021). Characterization of single-chain FV fragments of neutralizing antibodies to rabies virus glycoprotein. Viruses 13(11): 2311. https://doi.org/10.3390/v13112311

Zajac MD, Ortega MT, Moore SM. (2023). Development and evaluation of an Enzyme-Linked immunosorbent assay targeting Rabies-Specific IGM and IGG in human SERA. Viruses 15(4): 874. https://doi.org/10.3390/v15040874

Zhai L, Wang H, Zhao W, Zhang S, Miao F, Cao Y, Chen C, Li Y, Gao J, Lv R, Zhang S, Cao J, Zhang X, Yang M, Zhang B, Zhao J, Wei J, Gao J. (2022). Efficacy of ormutivimab, a novel recombinant human anti-rabies monoclonal antibody, in post-exposure prophylaxis animal models. Travel Medicine and Infectious Disease 46: 102267. https://doi.org/10.1016/j.tmaid.2022.102267

Zhang J, Shi N, Li G, Li L, Bai Y, Yang L, Zhao W, Gao J, Wei J, Zhao W, Zhai L, Huo P, Ren L, Yu L, Li Y. (2022). Population pharmacodynamic analyses of human anti-rabies virus monoclonal antibody (ormutivimab) in healthy adult subjects. Vaccines 10(8): 1218. https://doi.org/10.3390/vaccines10081218

Zorzan M, Castellan M, Gasparotto M, De Melo GD, Zecchin B, Leopardi S, Chen A, Rosato A, Angelini A, Bourhy H, Corti D, Cendron L, De Benedictis P. (2023). Antiviral mechanisms of two broad-spectrum monoclonal antibodies for rabies prophylaxis and therapy. Frontiers in Immunology 14: 1186063. https://doi.org/10.3389/fimmu.2023.1186063