Process for detergent-free production of outer membrane vesicles of a gram-negative bacterium
Inventors
Van De Waterbeemd, Bas • Van Der Pol, Leonardus Aldolfus
Assignees
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Publication Number
US-9272006-B2
Publication Date
2016-03-01
Expiration Date
Abstract
The present invention relates to the fields of medical microbiology and vaccines. In particular the invention relates to a process for detergent-free preparation of outer membrane vesicles (OMV) of Gram negative bacteria for use in vaccines, to OMV obtainable by said process, and to a pharmaceutical composition comprising such OMV. The present invention further relates to the use of OMV of the present invention as a medicament in particular for use in a method for eliciting an immune response.
Core Innovation
The invention relates to a process for producing a detergent-free preparation of bacterial outer membrane vesicles (OMV) for use in vaccines. The process cultivates a population of Gram-negative bacteria in culture to stationary growth phase, and after stationary growth phase onset uses the bacteria as the starting material for OMV extraction without using a detergent-based preparation approach.
The process begins extraction at a time at least about 1 hour after onset of the stationary growth phase. The incubation medium is adjusted to, or has, a pH higher than pH 8.0 and contains a concentration of a metal chelating agent between about 1 and 100 mM to extract the bacterial OMV.
After extraction, the process recovers the extracted bacterial OMV by separation of the bacterial OMV from the bacteria. The described framework supports downstream handling and formulation for vaccines or medicaments while maintaining the detergent-free process context.
Claims Coverage
The independent claim recites a detergent-free OMV production process for vaccine use based on three core inventive steps, with multiple dependent claims refining the extraction conditions, the chelating agent, bacterial genetic/phenotypic constraints tied to LPS toxicity/adjuvant activity, and optional formulation refinements as described in the provided partial content.
Detergent-free OMV production for vaccine use via stationary-phase incubation, metal chelator extraction, and OMV/bacteria separation
A process comprising cultivating a population of Gram-negative bacteria in culture to stationary growth phase; beginning at a time at least about 1 hour after onset of the stationary growth phase, incubating the bacteria obtained in step (a) in a medium adjusted to, or having a pH higher than, pH 8.0 and a concentration of a metal chelating agent of between about 1 and 100 mM to extract said bacterial OMV; and after step (b), recovering the extracted bacterial OMV by separation of the bacterial OMV from the bacteria.
Stationary-phase delayed extraction window
The process further defines beginning the incubating step at a time at least about 1 hour after onset of the stationary growth phase.
Extraction medium pH and metal chelating agent concentration
The process defines incubating in a medium adjusted to, or having, a pH higher than pH 8.0 and a concentration of a metal chelating agent of between about 1 and 100 mM to extract said bacterial OMV.
Chelating agent selection as EDTA
The chelating agent used is EDTA.
LPS with reduced toxicity while retaining at least part of LPS adjuvant activity
The Gram-negative bacteria produce an LPS with reduced toxicity versus corresponding wild-type while retaining at least part of LPS adjuvant activity by decreasing/knocking out lpxL1 (or a sequence ≥95% identical to SEQ ID NO:1), lpxL2 and/or lpxK and/or by altering lpxE and/or pagL expression.
Formulation with a pharmaceutically acceptable excipient
The process further includes combining the recovered bacterial OMV with a pharmaceutically acceptable excipient.
Across the provided independent claim and its supported refinements, claim coverage centers on a detergent-free OMV production process using stationary-phase Gram-negative bacteria, delayed extraction in a pH-above-pH-8.0 metal-chelator-containing medium, and recovery by separating OMV from bacteria, with optional refinements including specifying EDTA, constraining LPS toxicity/adjuvant activity through genetic modifications, and combining recovered OMV with a pharmaceutically acceptable excipient.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Use for vaccines.
Formulation for vaccines or medicaments.
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