Methods for the production of 3-o-deactivated-4'-monophosphoryl lipid a (3d-mla)

a technology of monophosphoryl lipid and production method, which is applied in the direction of antibody medical ingredients, drug compositions, immunological disorders, etc., to achieve the effect of simple and inexpensive steps and reduced phospholipid conten

Inactive Publication Date: 2007-09-13
CORIXA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0021] This method provides LPS solutions in CM that have reduced phospholipid content and that are therefore well-suited to further modification and purification to 3D-MLA. The method involves relatively simple and inexpensive steps.

Problems solved by technology

The fact that some of the responses are harmful, and some of these can be fatal, has precluded clinical use of LPS per se.
However, purification of LPS from the LPS- and phospholipid-rich CM phase typically requires multiple precipitation steps to obtain LPS of sufficient purity for use in immunostimulatory applications such as, for example, use as a vaccine adjuvant.

Method used

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  • Methods for the production of 3-o-deactivated-4'-monophosphoryl lipid a (3d-mla)

Examples

Experimental program
Comparison scheme
Effect test

example 1

General Methods

[0063] A. Media Preparation

[0064] Cell growth was conducted in M9 medium, which is prepared by combining sterile solutions of inorganic salts, casamino acids, and dextrose. The M9 salt solution is typically prepared in the fermentor and contains the following salts: 2.0 g / L NaCl, 0.2 g / L MgSO4.7H2O, 3.0 g / L KH2PO4, 6.0 g / L Na2HPO4, and 1.0 g / L NH4Cl. Sterile solutions of 20% (w / v) casamino acids (20 mL / L) and 50% (w / v) dextrose (32 mL / L) and then added aseptically to the fermentor to yield the completed medium.

[0065] B. Seed Growth

[0066] Typically, a sterile 250 mL Erlenmeyer flask was charged with 50 mL sterile M9 medium. A seed vial of Salmonella minnesota R595 (ca. 108 cfu) was thawed and added to the flask, which was then stoppered with a gauze plug. The culture was incubated at 37° C. for 6-8 h, until robust growth is evident.

[0067] C. Cell Growth

[0068] Cultures of Salmonella minnesota R595 were grown in a BioFlo III fermentor (New Brunswick Scientific, Inc...

example 2

Analytical Methods

[0075] A. Thin Layer Chromatography (TLC) of MLA and Related Samples

[0076] All TLC analyses were carried out using 5×10 cm plates coated with Silica Gel 60 (E Merck). Samples were generally applied to the TLC plates as 10 mg / mL solutions in chloroform:methanol 4:1 (v / v), with 3 μL solution (30 μg sample) applied in small spots to a 5 mm line using a capillary pipette. Plates were developed with a solvent system comprising chloroform / methanol / water / ammonium hydroxide 50:31:6:2 (v / v). Bands on the developed plates were visualized by spraying with a solution of 10% (w / v) phosphomolybdic acid in ethanol followed by charring at 150-160° C. In some cases, relative intensities of spots were quantified by scanning densitometry with a Shimadzu CS9000U Dual Wavelength Flying Spot Scanner (Shimadzu Corp.), using a scanning wavelength of 520 nm.

[0077] B. Analysis of MLA / 3D-MLA by High Performance Liquid Chromatography (HPLC)

[0078] Samples to be analyzed were first converte...

example 3

Comparison of Congener Composition of MLA / 3D-MLA from Cultures Harvested at Different Times

[0083] A series of fermentor runs was conducted with the following parameters: 2.0 L M9 medium (initial pH 6.84-6.87), 2 Lpm air flow, stirring at 50 rpm, 37° C., no pH control. Cultures were monitored by measuring optical density at 590 nm and were stopped when the desired growth stage was attained. Cells were processed and extracted as described above to yield LPS samples, which were then hydrolyzed to MLA and 3D-MLA and analyzed by HPLC (see Examples 1 and 2). Results are summarized in Table 1.

TABLE 1Congener composition of MLA and 3D-MLA from cells harvested at different ages.MLA3D-MLA3-O-3-O-Culture ageTime indeacylateddeacylatedRunDescriptionat harveststationary phasehexaacylheptaacylhexaacylALate exponential6.75 h N / A12.4%12.2%9.9%BEarly stationary9.5 h˜0.5 h9.2%6.8%9.2%phaseCLate stationary 15 h  ˜6 h19.5%13.2%21.5%phase

[0084] The data show that cultures of S. minnesota R595 alter t...

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Abstract

Herein is disclosed a method for producing lipopolysaccharide (LPS), comprising: (a) growing a culture of deep rough mutant bacterial strain in a medium; (b) maintaining the culture in stationary phase for at least about 5 hr; (c) harvesting cells from the culture; and (d) extracting LPS from the cells. The method allows for the production of an LPS which can be used to produce a 3-O-deacylated monophosphoryl lipid A (3D-MLA) having at least about 20 mol % of the hexaacyl congener group. Also herein is disclosed a method of extracting lipopolysaccharide (LPS) from a culture of deep rough mutant bacterial strain cells, comprising: (a) extracting the cells with a solution consisting essentially of at least about 75 wt % of an aliphatic alcohol having from 1 to 4 carbon atoms and the balance water, thereby producing cells with reduced phospholipid content; and (b) extracting the cells with reduced phospholipid content with a solution comprising chloroform and methanol, thereby yielding a solution of LPS in chloroform and methanol (CM). This method provides LPS solutions in CM that have reduced phospholipid content and are produced by relatively simple and inexpensive process steps.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application is a non-provisional of and claims the benefit of U.S. Provisional Application No. 60 / 280,089, filed Mar. 30, 2001.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] NOT APPLICABLE BACKGROUND OF THE INVENTION [0003] 1. Field of the Invention [0004] The present invention relates generally to the field of biosynthetic production of 3-O-deacylated-4′-monophosphoryl lipid A (3D-MLA). More particularly, it concerns methods of improving the yield of desired 3D-MLA congeners or minimizing the cost of purifying lipopolysaccharide (LPS) precursors of 3D-MLA. [0005] 2. Description of Related Art [0006] It has long been recognized that enterobacterial lipopolysaccharides (LPS) are potent stimulators of the immune system. A variety of responses, both beneficial and harmful, can be elicited by submicrogram amounts of LPS. The fact that some of the responses are harmful, and some of t...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P19/28C08B37/00C12N1/21C12P1/04A61K31/739A61P37/04C12N1/20C12P19/04
CPCA61K2039/55572C07H13/06C12P19/04A61P37/04C12N1/20
Inventor MYERS, KENT R.SNYDER, D. SCOTT
Owner CORIXA CORP
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