Effect of Washing Solutions on Diafiltration Efficiency and Properties of Klebsiella pneumoniae Antigenic Complexes

The prevalence and antibiotic resistance of ESKAPE bacteria cause difficulties in the prevention and treatment of their infections. Specialists of the Protective Antigen Laboratory of I.I. Mechnikov Scientific Research Institute of Vaccines and Serums continue work on  developing immunostimulating drugs for the prevention of diseases caused by some representatives of this group, including Klebsiella pneumoniae. When producing a drug and isolating a protective antigen, hydroxylamine is used as an inactivating agent. Purification of the final product from this toxic substance is complicated by a polysaccharide capsule secreted by K. pneumoniae. In this work, we evaluate the diafiltration efficiency of various washing solutions and their effect on the specific activity and chemical composition of the resulting antigen complexes of K. pneumoniae. To that end, a submerged cultured strain of K. pneumoniae 204 was used. The cells of the resulting culture were isolated by centrifugation and inactivated with hydroxylamine. The inactivating agent was removed by ultrafiltration in diafiltration mode using water or Tris-HCl buffers (pH=9.0) of  various concentrations. The as-obtained antigens were assessed in terms of the content of residual hydroxylamine, polysaccharides, protein by the Lowry method, nucleic acids by the Spirin method, and specific activity by hemagglutination inhibition assay. Samples of antigen-containing fluid of K. pneumoniae 204 were obtained. The use of Tris-HCl buffers (pH=9.0) for hydroxylamine removal led to a reduction in the hydroxylamine content to acceptable values (less than 1 μg/mL) when using a 25-fold volume of  the solution in any of the studied concentrations. At the same time, the use of distilled water as a purifying solution in  similar volumes did not produce a comparable result. The chemical composition of the resulting antigenic complexes was analyzed. The use of a Tris-HCl buffer solution (pH=9.0) at a concentration of 10 mM proves optimal for removing hydroxylamine from an antigen-containing fluid of K. pneumoniae, having no effect on the chemical composition and activity of its antigen complexes.

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