{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,31]],"date-time":"2026-10-31T03:31:59Z","timestamp":1793417519308,"version":"4.5.0"},"reference-count":31,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T00:00:00Z","timestamp":1784246400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Health of the Republic of Kazakhstan","award":["IRN BR25293294"],"award-info":[{"award-number":["IRN BR25293294"]}]},{"award":["IRN BR25293294"],"award-info":[{"award-number":["IRN BR25293294"]}],"id":[{"id":"https:\/\/ror.org\/022syee28","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Limited Liability Partnership Research Institute"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background: Brucellosis remains a significant zoonotic infection affecting approximately 500,000 people worldwide annually, with no licensed human vaccine available. Recombinant influenza virus vectors expressing Brucella spp. antigens represent a promising vaccine platform. However, transitioning from laboratory constructs to clinical candidates requires validation of scalable purification methods compliant with Good Manufacturing Practice (GMP) standards. This study aimed to develop and optimize a pilot-scale purification protocol for these vectors. Methods: Recombinant influenza A viruses (H5N1) expressing Brucella spp. antigens (Omp16, Omp19, L7\/L12, Cu-Zn SOD) were propagated in MDCK cell culture. The optimized purification process included: (1) clarification; (2) ultrafiltration\/diafiltration (100 kDa MWCO); (3) two-step chromatography (anion-exchange Q-Sepharose\u00ae Fast Flow and multimodal Capto\u2122 Core 700); and (4) sterile filtration. Process validation was performed across three independent pilot-scale batches (20 L each). Results: The purification process demonstrated high reproducibility for all constructs. Final preparations met established quality criteria: infectious titer \u2265 5.2 log10 TCID50\/mL, hemagglutination activity 7.33 \u00b1 0.58\u20138.33 \u00b1 0.58 log2, total protein content 157\u2013305 \u03bcg\/mL, residual host cell DNA &lt; 10 ng\/dose, and bacterial endotoxin levels \u2264 0.15 IU\/mL. The overall recovery of infectious virus was 20\u201324%, an optimal value for multi-stage bioprocessing. Preservation of the target genetic insert was confirmed in all final preparations by PCR and sequencing. Conclusions: The developed integrated purification protocol yields vectors with high purification efficiency, preserving biological activity and meeting regulatory quality requirements for residual host cell DNA and endotoxins. The technological platform demonstrated versatility, robustness (inter-batch coefficient of variation for yield did not exceed 10\u201312%), and scalability, establishing a foundation for preclinical and clinical studies of candidate brucellosis vaccines.<\/jats:p>","DOI":"10.3390\/vaccines14070626","type":"journal-article","created":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T09:00:52Z","timestamp":1784278852000},"page":"626","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Pilot-Scale Downstream Processing of Recombinant Influenza Virus Vectors Expressing Brucella spp. Antigens Using an Integrated Membrane-Chromatography Purification Platform"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7267-3931","authenticated-orcid":false,"given":"Nurika","family":"Assanzhanova","sequence":"first","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding \u201cQazBioPharm\u201d, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5938-3454","authenticated-orcid":false,"given":"Aigerim","family":"Sagymbayeva","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding \u201cQazBioPharm\u201d, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6976-1540","authenticated-orcid":false,"given":"Gaukhar","family":"Shynybekova","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding \u201cQazBioPharm\u201d, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8777-8453","authenticated-orcid":false,"given":"Kamshat","family":"Shorayeva","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding \u201cQazBioPharm\u201d, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9578-5140","authenticated-orcid":false,"given":"Sholpan","family":"Ryskeldinova","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding \u201cQazBioPharm\u201d, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4722-0495","authenticated-orcid":false,"given":"Aigerim","family":"Mailybayeva","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding \u201cQazBioPharm\u201d, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0627-0442","authenticated-orcid":false,"given":"Yeldos","family":"Myrzakhmetov","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding \u201cQazBioPharm\u201d, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-5245-4444","authenticated-orcid":false,"given":"Ekaterina","family":"Yamanova","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding \u201cQazBioPharm\u201d, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-6131-6470","authenticated-orcid":false,"given":"Rassul","family":"Sidikhov","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding \u201cQazBioPharm\u201d, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0450-5941","authenticated-orcid":false,"given":"Meiyrim","family":"Almezhanova","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding \u201cQazBioPharm\u201d, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-3403-6268","authenticated-orcid":false,"given":"Samat","family":"Zhaksylyk","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding \u201cQazBioPharm\u201d, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0510-5496","authenticated-orcid":false,"given":"Zharkynai","family":"Absatova","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding \u201cQazBioPharm\u201d, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4238-5116","authenticated-orcid":false,"given":"Kuandyk","family":"Zhugunissov","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding \u201cQazBioPharm\u201d, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7954-6246","authenticated-orcid":false,"given":"Olga","family":"Chervyakova","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding \u201cQazBioPharm\u201d, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8896-3482","authenticated-orcid":false,"given":"Nurlan","family":"Akmyrzayev","sequence":"additional","affiliation":[{"name":"Research Institute for Biological Safety Problems, National Holding \u201cQazBioPharm\u201d, Gvardeiskiy 080409, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2325","DOI":"10.1056\/NEJMra050570","article-title":"Brucellosis","volume":"352","author":"Pappas","year":"2005","journal-title":"N. Engl. J. Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/S1473-3099(06)70382-6","article-title":"The new global map of human brucellosis","volume":"6","author":"Pappas","year":"2006","journal-title":"Lancet Infect. Dis."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Dean, A.S., Crump, L., Greter, H., Schelling, E., and Zinsstag, J. (2012). Global burden of human brucellosis: A systematic review of disease frequency. PLoS Negl. Trop. Dis., 6.","DOI":"10.1371\/journal.pntd.0001865"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1016\/S1473-3099(07)70286-4","article-title":"Human brucellosis","volume":"7","author":"Franco","year":"2007","journal-title":"Lancet Infect. Dis."},{"key":"ref_5","unstructured":"Ivanov, N.P. (2007). Brucellosis in Animals and Control Measures, Atamura. (In Russian)."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e29026","DOI":"10.1016\/j.heliyon.2024.e29026","article-title":"The spatiotemporal epidemiological study on human brucellosis in Shenyang, China from 2013 to 2022","volume":"10","author":"Chen","year":"2024","journal-title":"Heliyon"},{"key":"ref_7","first-page":"233","article-title":"Brucellosis in humans\u2014Etiology, diagnostics, clinical forms","volume":"20","year":"2013","journal-title":"Ann. Agric. Environ. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1016\/j.trstmh.2007.02.027","article-title":"Brucellosis as a cause of acute febrile illness in Egypt","volume":"101","author":"Jennings","year":"2007","journal-title":"Trans. R. Soc. Trop. Med. Hyg."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Elbehiry, A., Aldubaib, M., Marzouk, E., Abalkhail, A., Almuzaini, A.M., Rawway, M., Alghamdi, A., Alqarni, A., Aldawsari, M., and Draz, A. (2023). The Development of Diagnostic and Vaccine Strategies for Early Detection and Control of Human Brucellosis, Particularly in Endemic Areas. Vaccines, 11.","DOI":"10.3390\/vaccines11030654"},{"key":"ref_10","first-page":"867","article-title":"Human health benefits from livestock vaccination for brucellosis: Case study","volume":"81","author":"Roth","year":"2003","journal-title":"Bull. World Health Organ."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1146\/annurev-micro-090110-102905","article-title":"Interactions of the human pathogenic Brucella species with their hosts","volume":"65","author":"Atluri","year":"2011","journal-title":"Annu. Rev. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Assanzhanova, N., Zhugunissov, K., Chervyakova, O., Ryskeldinova, S., Akmyrzayev, N., Sagymbayeva, A., Myrzakhmetov, Y., and Mailybayeva, A. (2026). Modern Strategies for Brucellosis Vaccination: From Traditional Approaches to Innovative Platforms. Vaccines, 14.","DOI":"10.3390\/vaccines14050409"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Gonz\u00e1lez, D., Grill\u00f3, M.J., De Miguel, M.J., Ali, T., Arce-Gorvel, V., Delrue, R.M., Conde-Alvarez, R., Mu\u00f1oz, P., L\u00f3pez-Go\u00f1i, I., and Iriarte, M. (2008). Brucellosis vaccines: Assessment of Brucella melitensis lipopolysaccharide rough mutants defective in core and O-polysaccharide synthesis and export. PLoS ONE, 3.","DOI":"10.1371\/journal.pone.0002760"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1505","DOI":"10.1016\/j.ajpath.2015.03.003","article-title":"Pathogenesis and immunobiology of brucellosis: Review of Brucella\u2013host interactions","volume":"185","author":"Ficht","year":"2015","journal-title":"Am. J. Pathol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Bugybayeva, D., Ryskeldinova, S., Zinina, N., Sarmykova, M., Assanzhanova, N., Kydyrbayev, Z., and Tabynov, K. (2020). Development of human vectored brucellosis vaccine formulation: Assessment of safety and protectiveness of influenza viral vectors expressing Brucella immunodominant proteins in mice and guinea pigs. BioMed Res. Int., 2020.","DOI":"10.1155\/2020\/1438928"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1385\/MB:22:1:087","article-title":"Downstream processing in the biotechnology industry","volume":"22","author":"Kalyanpur","year":"2002","journal-title":"Mol. Biotechnol."},{"key":"ref_17","unstructured":"World Health Organization (2013). Annex 4: Recommendations to Assure the Quality, Safety and Efficacy of Influenza Vaccines (Human, Live Attenuated) for Intranasal Administration, WHO. WHO Technical Report Series No. 977."},{"key":"ref_18","unstructured":"European Medicines Agency Guideline on Quality, Non-Clinical and Clinical Aspects of Live Recombinant Viral Vectored Vaccines; EMA\/CHMP\/VWP\/141697\/2009, EMA. Available online: https:\/\/www.ema.europa.eu\/en\/documents\/scientific-guideline\/guideline-quality-non-clinical-and-clinical-aspects-live-recombinant-viral-vectored-vaccines_en.pdf."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1016\/j.virol.2012.09.019","article-title":"Monolithic ion exchange chromatographic methods for virus purification","volume":"434","author":"Oksanen","year":"2012","journal-title":"Virology"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2487","DOI":"10.1016\/j.vaccine.2014.02.086","article-title":"Purification of Vero cell derived live replication deficient influenza A and B virus by ion exchange monolith chromatography","volume":"32","author":"Banjac","year":"2014","journal-title":"Vaccine"},{"key":"ref_21","unstructured":"Weigel, T. (2022). Development of Chromatography-Based Purification Processes for Cell Culture-Derived Influenza Virus Particles. [Doctoral Dissertation, der Otto-von-Guericke-Universit\u00e4t]."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Mailybayeva, A., Yespembetov, B., Ryskeldinova, S., Zinina, N., Sansyzbay, A., Renukaradhya, G.J., and Tabynov, K. (2017). Improvedinfluenza viral vector-based Brucella abortus vaccine induces robust B and T-cell responses and protection against Brucella melitensisinfection in pregnant sheep and goats. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0186484"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1093\/oxfordjournals.aje.a118408","article-title":"A simple method of estimating fifty per cent endpoints","volume":"27","author":"Reed","year":"1938","journal-title":"Am. J. Epidemiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/S0021-9258(19)52451-6","article-title":"Protein measurement with the Folin phenol reagent","volume":"193","author":"Lowry","year":"1951","journal-title":"J. Biol. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1007\/s40259-017-0234-5","article-title":"Adeno-Associated Virus (AAV) as a Vector for Gene Therapy","volume":"31","author":"Naso","year":"2017","journal-title":"BioDrugs"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e3055","DOI":"10.1002\/btpr.3055","article-title":"High throughput screening setup of a scale-down device for membrane chromatography-aggregate removal of monoclonal antibodies","volume":"36","author":"Stein","year":"2020","journal-title":"Biotechnol. Prog."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"100211","DOI":"10.1016\/j.dche.2024.100211","article-title":"Controlling tangential flow filtration in biomanufacturing processes via machine learning: A literature review","volume":"14","author":"Oetomo","year":"2025","journal-title":"Digit. Chem. Eng."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"767","DOI":"10.1002\/jssc.200401812","article-title":"Monoliths for fast bioseparation and bioconversion and their applications in biotechnology","volume":"27","author":"Jungbauer","year":"2004","journal-title":"J. Sep. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"100780","DOI":"10.1016\/j.coche.2021.100780","article-title":"Challenges in downstream purification of gene therapy viral vectors","volume":"35","author":"Singh","year":"2022","journal-title":"Curr. Opin. Chem. Eng."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"237","DOI":"10.4155\/pbp.14.15","article-title":"Manufacturing of viral vectors: Part II. Downstream processing and safety aspects","volume":"2","author":"Kamen","year":"2014","journal-title":"Pharm. Bioprocess."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Pan, Y., Yan, J., Zhang, Y., Lin, J., Liang, Z., and Sun, J. (2025). Centrifugation-Based Purification Protocol Optimization Enhances Structural Preservation of Nucleopolyhedrovirus Budded Virion Envelopes. Insects, 16.","DOI":"10.3390\/insects16040424"}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/7\/626\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T08:21:33Z","timestamp":1786090893000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/7\/626"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,17]]},"references-count":31,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["vaccines14070626"],"URL":"https:\/\/doi.org\/10.3390\/vaccines14070626","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,17]]}}}