Purification of biological preparations

Details Purification of biological preparations Purification of biological preparations

2000-04-03

2001-10-30

Low, Christopher S. F. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

Separation or purification

C530S415000, C530S417000, C530S351000, C530S371000, C536S025400, C536S023100, C536S123100, C210S198200, C210S651000

Reexamination Certificate

active

06310186

ABSTRACT:

PRIORITY DATA
This application claims the benefit of foreign priority under 35 U.S.C. §119(
a
)-(
d
) from Great Britain Application No. 990753.3, filed Apr. 1, 1999, herein incorporated by reference.
This invention relates to the purification of biological preparations such as proteins and nucleic acids, especially for example proteins that have been produced by recombinant DNA techniques in bacteria. In a particular embodiment, the invention concerns improved methods for reducing the content of contaminants in biological preparations, e.g. recombinant proteins produced in host bacteria.
BACKGROUND OF THE INVENTION AND PRIOR ART
It is known to produce recombinant proteins in gram negative host bacteria, for example
E.coli,
Pseudomonas, Vibrio spp. and
Methylophilus methylotrophus.
One difficulty that can be encountered in this approach is that
E.coli,
and other such gram negative host bacteria useful for the expression of heterologous proteins are able to produce bacterial toxins such as endotoxins, which are integral lipo-polysaccharide (LPS) components of the bacterial cell wall, and which can be highly pyrogenic and can cause febrile reactions in animals.
It is desirable to reduce contaminating endotoxins in biological preparations when they are encountered.
There are known bioassays to detect bacterial endotoxin contamination of biological materials: the test used is the Chromagenic peptide endpoint method and this test has been adopted by the European Pharmacopoeia Commission and is described in 1999 European Pharmacopoeia—Supplement 1999, and can be used when needed for the purposes of the present invention.
Certain methods intended to remove or reduce levels of contaminating endotoxin from biological materials e.g. proteins are already known in the art.
Anion exchange chromatography for endotoxin removal is discussed by K. Khandake in an article entitled “Effective Removal of Negatively Charged Interfering Moleculed from Proteins” (U.S. Bulletin 2204, a technical bulletin from BioRad lnc., N.J., U.S.A.). Such chromatography is also discussed in “Why Anion Exchange Works So Well for Endotoxin Removal . . . Sometimes”, (P. Gagnon, in Summer 1998 issue of “Validated Biosystems Quarterly Resource Guide for Downstream Processing, Validated Biosystems Inc., Tucson, Ariz., U.S.A.) K. C. Hou and R. Zaniewski, in Biotechnology and Applied Biochemistry 12, 315-324, 1990, describe endotoxin separation from albumin and gamma-globulin solutions using anion-exchange polymeric matrices carrying DEAE or QAE functional groups.
T. E. Karplus et al., in Journal of Immunological methods, 105, 211-220, 1987, describe the use of affinity-binding using polymyxin B-Sepharose (TM) in a method for reducing endotoxin contamination in catalase and IgG solutions.
K. W. Talmadge and C. J. Siebert, in Journal of Chromatography, 476, 175-185, 1989, report separation of endotoxin from serum albumin and IgG using a polymyxin-derivatised macroporous polymer affinity column.
F. B. Anspach and O. Hilbeck, in Journal of Chromatography A, 711, 81-92, 1995, describe the use of histidine, histamine and polymyxin B affinity sorbents in separation of
E. coli
-derived endotoxin from serum albumin and lysozyme. The authors concluded that “an endotoxin-specific sorbent for general decontamination of protein solutions seems not to be available”.
The present inventors consider that endotoxin removal from biological materials remains a problem and that there remains a need for further techniques for endotoxin removal. Accordingly, an aim of the present invention is to provide new purification procedures for the reduction of endotoxin contamination e.g. associated with recombinant nucleic acids, or with recombinant proteins, for example proteins produced in host bacteria.
SUMMARY AND DESCRIPTION OF THE INVENTION
According to an aspect of the invention we provide a method for separating bacterial endotoxin associated as a contaminant with biological material which comprises, preferentially adsorbing the endotoxin onto a solid phase by contacting the biological material with a hydrophobic solid phase in the presence of a charged solubilising agent and a water soluble salt.
This process is particularly applicable under conditions where the biological material to be decontaminated is less hydrophobic than the endotoxin, and this can be assessed, e.g. as described below.
To determine whether endotoxin is less hydrophobic than the biological material under test, the unbound material obtained from contacting the biological material with the solid phase, can be tested for either the presence of endotoxin, or it can be tested for the presence of the biological material. Alternatively, the unbound material can be tested to determine levels of both endotoxin and biological material. When the unbound material from the test is found to contain substantially more of the wanted biological material than of the endotoxin this indicates that the wanted biological material is less hydrophobic than the endotoxin, and vice versa.
For the purpose of carrying out such testing, bacterial endotoxin contamination of biological material, e.g. protein can be determined using the Chromagenic peptide endpoint method described in 1999 European Pharmacopoeia—Supplement 1999. When the biological material is a protein or a peptide it can be detected for example, by Western blotting of an eluate sample. When the biological material is DNA it can be detected for example, by Southern blotting of an eluate sample. When the biological material is RNA it can be detected for example, by Northern blotting of an eluate sample.
The biological material which it can be desired to separate from associated endotoxin can be for example, a protein or a peptide susceptible to endotoxin contamination. For example, certain proteins may be found to be contaminated to unacceptable levels by endotoxin when expressed in the form of inclusion bodies in an
E.coli
heterologous expression system, and when the inclusion bodies are solubilised in standard salt or urea solutions.
Thus, examples of the process according to the invention can be used in the purification of protein or peptide susceptible to endotoxin contamination, e.g. because they can bind with endotoxin.
Certain examples of the process can be used for purification of recombinant proteins, for example a viral subunit antigen for vaccine use. The viral antigen can be for example, an antigen of papillomavirus, e.g. L2, E6 or E7, or a fusion protein involving one or more of these antigens e.g. as described in WO 96/26277 (Cantab Pharmaceuticals Research Limited, Whittle et al.).
Otherwise, the biological material which it is desirable to separate from endotoxin can be for example, a nucleic acid susceptible to endotoxin contamination. The nucleic acid can be DNA or RNA for use, e.g. as a vaccine, or for gene therapy.
The endotoxin which it is desirable to separate from associated biological material can be for example, lipo-polysaccharide from gram negative bacteria, e.g.
E.coli.
The hydrophobic solid phase can be for example a derivatised particulate material with a particle or bead size suitable for use as a column chromatography matrix, or alternatively it can be a derivatised sheet material, e.g. in the form of a membrane.
The hydrophobic particulate or sheet material can be a hydrophilic base material that has been derivatised with hydrophobic groups.
The hydrophilic base material can be, for example, a polysaccharide, e.g. agarose or dextran, or alternatively it can be, for example, a polyamide, e.g. acrylamide. Alternatively, the hydrophobic matrix can be either a non-derivatised or derivatised hydrophobic base matrix, for example, a polystyrene di-vinyl benzene co-polymer base matrix, e.g. Source (TM) matrix, available from Pharmacia, or Poros (TM) available from PerSeptive Biosystems.
The hydrophobic groups used to derivatise the base matrix can be for example aromatic groups, e.g. phenyl groups, e.g. as in Phenyl Sepharose (TM) (Pharmacia). Alternatively, the hydrophobic derivatives can be alkyl g

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