mesЭкстремальная биомедицинаExtreme Medicine3033-89643033-8972Centre for Strategic Planning of the Federal Medical and Biological Agency10.47183/mes.2021.029mes-146Research ArticleОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕORIGINAL RESEARCHСравнение методов очистки фаговых лизатов грамотрицательных бактерий для персонализированной терапииComparison of methods for purification of bacteriophage lysates of gram-negative bacteria for personalized therapyГородничевР. Б.GorodnichevR. B.

Роман Борисович Городничев

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Roman B. Gorodnichev

Malaya Pirogovskaya, 1a, Moscow, 119435

gorodnichev.r.b@gmail.comКорниенкоМ. А.KornienkoM. A.

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Malaya Pirogovskaya, 1a, Moscow, 119435

КупцовН. С.KuptsovN. S.

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Malaya Pirogovskaya, 1a, Moscow, 119435

ЕфимовА. Д.EfimovA. D.

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Malaya Pirogovskaya, 1a, Moscow, 119435

БогданВ. И.BogdanV. I

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Malaya Pirogovskaya, 1a, Moscow, 119435

ЛетаровА. В.LetarovA. V.

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Malaya Pirogovskaya, 1a, Moscow, 119435

ШитиковЕ. А.ShitikovE. A.

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Malaya Pirogovskaya, 1a, Moscow, 119435

ИльинаЕ. Н.IlinaE. N.

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Malaya Pirogovskaya, 1a, Moscow, 119435

Федеральный научно-клинический центр физико-химической медицины Федерального медико-биологического агентстваРоссияFederal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological AgencyRussian FederationФедеральный исследовательский центр биотехнологииРоссияFederal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological AgencyRussian FederationФедеральный исследовательский центр биотехнологииРоссияFederal Research Center of BiotechnologyRussian Federation2021261020242333037Copyright © Городничев Р.Б., Корниенко М.А., Купцов Н.С., Ефимов А.Д., Богдан В.И., Летаров А.В., Шитиков Е.А., Ильина Е.Н., 20242024Городничев Р.Б., Корниенко М.А., Купцов Н.С., Ефимов А.Д., Богдан В.И., Летаров А.В., Шитиков Е.А., Ильина Е.Н.Gorodnichev R.B., Kornienko M.A., Kuptsov N.S., Efimov A.D., Bogdan V.I., Letarov A.V., Shitikov E.A., Ilina E.N.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.extrememedicine.ru/jour/article/view/146

Фаготерапия является перспективным методом лечения инфекций, вызванных устойчивыми к антибактериальным препаратам бактериями. Для получения безопасных терапевтических препаратов бактериофагов необходима глубокая очистка лизатов от компонентов бактериальной клетки, в частности эндотоксинов. Целью работы было исследовать применимость различных методов очистки фаголизатов для получения терапевтических препаратов. Фаги vB_KpnM_Seu621 (Myoviridae) и vB_KpnP_Dlv622 (Autographiviridae) использовали для получения лизата штамма Klebsiella pneumoniae KP9068. Очистку лизатов проводили методом осаждения бактериофагов с использованием полиэтиленгликоля, ультрацентрифугированием в градиенте CsCl, ультрацентрифугированием в градиенте сахарозы, с помощью центрифужных концентраторов (100 кДа), концентрированием на целлюлозных фильтрах 0,22 мкм в присутствии MgSO4. Уровень эндотоксинов определяли ЛАЛ-тестированием. В результате действия vB_KpnM_Seu621 и vB_KpnP_Dlv622 были получены лизаты с титром 1,25 × 1012 ± 7,46 × 1010 и 2,25 × 1012 ± 1,34 × 1011 БОЕ/мл и концентрацией эндотоксина 3806056 ± 429410 и 189456 ± 12406 ЕЭ/мл соответственно. Из традиционных методов оптимальным по снижению уровня эндотоксина и сохранению концентрации фаговых частиц было ультрацентрифугирование в градиенте CsCl (303 ± 20 – 313 ± 35 ЕЭ/мл, 1,5‒2,75 × 1012 ± 1,71 × 1011 БОЕ/мл); из альтернативных — очистка в градиенте сахарозы и фильтрация в присутствии MgSO4. Метод очистки лизатов следует подбирать для каждого препарата бактериофагов отдельно. Из способов очистки лизатов, пригодных для внутривенного и интратекального введения, перспективны метод ультрацентрифугирования в градиенте сахарозы и фильтрация в присутствии MgSO4.

Phage therapy is a promising method of treating antibiotic-resistant infections. To obtain a safe therapeutic formulation, bacterial cell components, including endotoxins, must be removed from the phage lysate. This study was aimed at comparing the efficacy of purification methods for phage lysates intended for therapeutic use. Phages vB_KpnM_Seu621 (Myoviridae) and vB_KpnP_Dlv622 (Autographiviridae) were grown using the KP9068 strain of Klebsiella pneumoniae as a host. The obtained lysates were purified using phage precipitation with polyethylene glycol, CsCl density gradient ultracentrifugation, sucrose density gradient ultracentrifugation, precipitation with 100 kDa centrifugal filter units, and phage concentration on 0.22 μm cellulose filters in the presence of MgSO4. Endotoxin concentrations were determined by LAL testing. The obtained lysates contained 1.25 × 1012 ± 7.46 × 1010 and 2.25 × 1012 ± 1.34 × 1011 PFU/ml of vB_KpnM_Seu621 and vB_KpnP_Dlv622, respectively, and had endotoxin concentrations of 3,806,056 ± 429,410 and 189,456 ± 12,406 EU/ml, respectively. CsCl gradient ultracentrifugation was found to be the optimal conventional purification method in terms of reducing endotoxin concentrations and maintaining phage titers (303 ± 20 — 313 ± 35 EU/ml, 1.5–2.75 × 1012 ± 1.71 × 1011 PFU/ml). Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 were found to be the optimal non-traditional purification methods. A method for phage lysate purification should be selected for each phage preparation individually. Sucrose gradient ultracentrifugation and filtration in the presence of MgSO4 hold promise as purification methods that can produce phage preparations suitable for intravenous administration.

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The authors declare that there are no conflicts of interest present.