WO1999019345A1

WO1999019345A1 - Method for purifying retroviral particles and soluble viral antigens

Info

Publication number: WO1999019345A1
Application number: PCT/US1998/021737
Authority: WO
Inventors: Yichen Lu
Original assignee: Institute For Vaccine Development; Avant Immunotherapeutics, Inc.
Priority date: 1997-10-14
Filing date: 1998-10-14
Publication date: 1999-04-22
Also published as: AU9695698A

Abstract

The present invention provides a method of purifying viral particles, preferably retroviral particles, and also soluble viral antigens. The present invention is based on the surprising discovery that by varying the conditions one can use hydrophobic interaction chromatography (HIC) alone, without additional chromatography steps, to purify retroviral particles and soluble retroviral antigens in a conformation that is substantially identical to the native forms.

Description

METHOD FOR PURIFYING RETROVIRAL PARTICLES AND SOLUBLE

VIRAL ANTIGENS

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of protein purification and more particularly to the purification of retroviral particles and soluble viral antigens.

2. Background

Protein purification involves a variety of competing demands - the ability to rapidly purify the protein to a desired purity, the desire to keep the protein in its proper conformational state and the desire to do so simply and effectively.

Consequently, purification procedures that are appropriate for research amounts of a protein frequently are ineffective or prohibitive in cost at the larger-scale necessary.

Much attention has focused on the use of viral vectors for use in gene transfer. One preferred group of viral vectors is retroviral vectors because of their efficiency in infecting cells. Presently, a great deal of interest has focused on the use of HIV-based vectors, which contain an HIV capsid. Attention has focused on the use of both those vectors containing an HIV envelope and a psuedotype envelope.

.Although work is ongoing on with these vectors in early stage clinical trials, attention is also being focused on the ability to create large-scale producer cells to produce large amounts of such vectors. In order to be able to use such viral vectors an efficient means for purifying the viral vectors particles is needed. Attention has also focused on the use of viral particles, particularly retroviral particles such as primate lentivirus particles in generating immune reactions. For example, there are currently clinical trails being conducted where particles comprising an HIV capsid and an HIV envelope produced by viral vectors are being injected into individuals to study their immune reaction. These particles can be produced by a variety of methods. However, the ability to purify them simply and efficiently is important.

The traditional method for concentrating and purifying such retroviral particles, e.g., HIV, has been through the use of multiple ultra- centrifugation steps. However, centrifugation results in significant loss of the envelope glycoprotein gp-120 and a subsequent loss of infectivity. In fact, centrifugation can result in as much as a 99.9% loss in the viral titer. Accordingly, there is a need for a more efficient method of retroviral particle purification that preserves infectivity, yet retains the particle in a conformationally appropriate form.

SUMMARY OF THE INVENTION The present invention provides a method of purifying viral particles, preferably retroviral particles, and also soluble viral antigens.

The present invention is based on the surprising discovery that by varying the conditions one can use hydrophobic interaction chromatography (HIC) alone, without a chromotography step such as ion exchange chromatography, to purify retroviral particles and soluble retroviral antigens in a conformation that is substantially identical to the native forms.

Preferred retroviral particles and soluble antigens are retroviral gene delivery vectors such as Moloney Leukemia Virus, HIV and immunogenic viral vectors and antigens. Move preferably, the use of retroviruses from the lentiviruses subfamily and include, for example primate lentiviruses, HIV-1, HIV-2 and SIV. HIV-1 and HIV-2 are preferred. HIV-1 is more preferred. The retroviral particles and soluble antigens may be isolated from a native source or may be obtained by recombinant means. Retroviral particles include both infectious and non-infectious, e.g., empty viral capsids and pseudotyped particles.

Soluble antigens include gp- 120, p24, gp41, pl7and p55.

DETAILED DESCRIPTION OF THE INVENTION

Hydrophobic interaction chromatography (HIC) is a purification techniques, in which the passage of molecules through a column is retarded by hydrophobic interactions between the column support material (or substance bound by the support material) and the molecules being fractionated.

Typical of such fractionating processes are high performance liquid chromatography processes using a hydrophobic column. A typical column is an ether-HIC or phenyl-HIC column. An ether-HIC column contains aliphatic groups linked to a column support material by an ether linkage, while a phenyl-HIC column contains phenyl groups linked to the support material. Those of ordinary skill in the art are aware of the basic HIC techniques, e.g., addition of sample to the column and elution carried out using solutions having sufficient ionic strength (which may for some molecules be zero) to cause the material being separated to "stick" to the surfaces of the resin used in the column. Lowering the ionic strength of the eluent (i.e., decreasing the concentration of salts in the eluent) reduces the tendency of hydrophobic materials to be retained by the column.

A variety of hydrophobic interaction chromatography resins can be used, and the present invention is not limited to any particular resin. Example of typical HIC columns include butyl (butyl Sepharose 4 fast (BS4) Parmacia), octyl (octyl Sepharose 4 fast (OS4), Pharmacia) and Phenyl (Phenyl Sepharose (PSH, PS6- 1 and PS6-h), Pharmacia). We have found that separation based on hydrophobic interactions does not appear to adversely affect the confirmation of the proteins being purified. This is particularly important with respect to "particles." Significantly, we were able to purify the entire viron (e.g. particle) along with the associated envelope glycoproteins, gp 41 and gp 120. Thus the multiple steps of ultra-centrifugation currently being used can be avoided.

One can also use the procedure to purify desired proteins such as soluble viral antigens (SVAs). These include envelope and capsid proteins, e.g., HIV gpl20, p55, p24.

Conditions under which these columns are used vary depending upon specific columns and with the protein(s) intended to be purified. Typical conditions include a pH of from about 6 to about 8.5; an ionic strength of from about 0.05 to 4.0M (expressed as NaCl) preferably about OJM to 2.0M; and elution using a gradient from 40% ammonium sulfate (or a different initial concentration as described above) decreasing to 0% ammonium sulfate. Preferred ionic strength with a phenyl based column is about 1.5 to 2.5M for NaCl, with about 2.0M being most preferred; about 0.5 to 1.5M for ammonium sulfate, with about 0.8M being most preferred; and about .25 to .75M for ammonium acetate, with about 0.4M being most preferred.

The present invention is based on the additional surprising discovery by the present inventor that under specific conditions, hydrophobic interaction chromatography (HIC) , without additional chromatography steps such as ion exchange chromatography and/ or gel filtration chromatography, can be used to produce a purified retroviral particle such as a HIV particle having its associated envelope, e.g. gp- 120, intact. It was further discovered that under specified conditions soluble SVAs such as HIV antigens, including, for example, gp-120 and p-24 could be purified using HIC. The retroviral particles and soluble retroviral antigens produced by the method of the present invention are substantially identical to the native forms in conformation.

This method can be used with any virus or viral vector based upon a virus that forms a particle, preferably associated with an envelope protein. Preferably, the virus is a RNA virus. More preferably a retrovirus. Still more preferably a lentivirus. Even more preferably a primate lentivirus. Most preferably HIV-1 or HIV-2. With viral particles this invention also can be used with pseudotyped particles.

The process of the present invention begins with a retroviral particle and/or soluble antigen source. Such a source can be a cell medium into which the particles or soluble antigens have been secreted or a cell or viral lysate. Both recombinant and mature viral sources may be used.

Retroviral particles and/ or soluble antigens can be obtained from any transfected cell. Preferred cells include COS or CHO cells.

In general the purification process of the present invention comprises, if necessary, separating the culture supernatant from cells using, for example, low speed centrifugation (about 1000 rpm); mixing the obtained solution with a preselected salt concentration and; fractionating the solution using at least one HIC step to provide the desired purified protein.

The preselected salt concentration and pH is based on the protein (particle and/ or soluble antigen) to be purified and the column selected. Representative conditions are set forth in the examples below.

Typically one condition will be optimal for purification of a particular protein, e.g., an envelope protein such as gpl20, and another condition optimal for a second viral protein such as a capsid protein, e.g., p55. However, we have found that by selecting less optimal but overlapping conditions we have been able to purify multiple proteins in a single step.

A single HIC step is preferred, but additional HIC steps can be used. In certain applications, if more than a single HIC step is used it may be desirable to use different HIC supports (e.g., separation on a phenyl-HIC column followed by separation on an ether-HIC column) . However, two separations on the same column (e.g., a phenyl-HIC column) can be used. Conditions can be adjusted using known techniques to provide for separation of peaks of protein having the desired activity. Fractions containing the desired activity are collected and separated from fractions not containing such activity.

The present invention is further illustrated by the following

Examples. These Examples are provided to aid in the understanding of the invention and are not construed as a limitation thereof.

Examples

The culture supernatant (CS) of CEM-174 cells chronically infected with a molecularly cloned HIV- 1 virus was used as a source of infectious virions and soluble viral antigens.

The HIV-1 virus corresponded to an HIV-1 genome wherein the 3' LTR was replaced by a tk HSV polyadenylation sequence, the integrase and reverse transcriptase genes were deleted and a cytomegalovirus promoter has been substituted from the NRE portion of the 5' LTR.

The CS was separated from cells by low speed centrifugation (lOOOrpm) and mixed with the stock solutions of different salt at various pH. Followed by passing the CS/salt solution through a 0.25 μm filter unit, 10 ml CS/salt was loaded onto a BS4 mini column previously washed with the salt solution that matched the sample. The HIC column was then washed with at least 50 ml of the same salt solution without the CS. The elution of the binding material was carried out with 1 ml PBS (pH 7). The level of HIV- 1 p24 and gpl20 antigens were then determined with the antigen-capture ELISA, respectively, and compared with that of the starting CS:

The recovery of HIV- 1 p24 antigens from HIC: Salt solution 2M 1M 0.8M 0.4M 0.2M

NaCl 39% 29% 1% (NH₄)₂SO₄ 38% 46% 71% 98% 78%*

NH₄Acetate 82% 61% 17%

PBS 0.3% positive control

*Starting CS is 100% The recovery of HIV- 1 gρl20 antigens from HIC: salt solution 0.8M 0.4M 0.2M

(NH₄)₂SO₄ ud ud ud

NH₄Acetate ud 80% ud PBS undetectable (ud)

It was concluded that 0.4 M NH₄Acetate at pH 7.0 was the optimal condition at which both HIV-1 p24 and gρl20 binds to BS4 column. This conclusion is further confirmed by FPLC analysis.

Using a Superdex 200 FPLC column for gel filtration of proteins, the material eluded from the aforementioned HIC column can be divided into 3 groups of proteins according their molecular weights (group A, B, C.). Group A represents proteins that have molecular weight much higher than 200 kd. They tested positive for both HIV-1 ρ24 (24 kd) and gpl20 (120 kd), indicating that this group represents HIV-1 virion. In fact, when proteins from group A were analyzed in a SDS PAGE, which should dissociate subunit HIV- 1 proteins from the virion, multiple protein bands were observed ranging from 14 kd to 160 kd. Moreover, many of these proteins can be recognized in a western blot analysis with serum from HIV- 1 infected patient, thus being consistence with the positive test result of p24 and gpl20 assays.

Group B contains proteins that have molecular weight from 50 kd to 160 kd. This group was tested positive for HIV-1 gpl20 by both CD4 capture ELISA and western analysis using HIV- 1 patient serum.

Group C contains proteins of 10-50 kd. HIV- 1 p24 protein can be detected by both antigen capture ELISA and western analysis using HIV- 1 patient serum.

In summary, this novel method can be used in large-scale preparation of HIV-1 virus and soluble antigens as an AIDS vaccine. The invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of this disclosure, may make modifications and improvements within the spirit and scope of the invention.

Claims

What is claimed is:

1. A process for purifying viral particles and/ or soluble viral antigens comprising loading a mixture containing viral particles and/ or soluble viral antigens onto a hydrophobic interaction chromatography column and eluting the viral particles and/ or soluble viral antigens from the column, wherein the process uses no ion exchange chromatography step.

2. The process of claim 1, wherein the viral particle and/ or soluble viral antigens are from retroviruses.

3. The process of claim 2, wherein the retroviral particles and/ or soluble antigens are from the lentiviruses subfamily.

4. The process of claim 3, wherein the viral particles and/or soluble antigens from the lentiviruses subfamily are from HIV-1, HIV-2 or SIV.

5. The process of claim 1, wherein the viral particles are infectious.

6. The process of claim 1, wherein the viral particles are non- infectious.

7. The process of claim 1, wherein the viral particles are pseudotyped particles.

8. The process of claim 1, wherein the soluble antigens are selected from the group of HIV proteins consisting of gp- 120, p24, gp41, pl7 and p55.

9. The process of claim 1, wherein the hydrophobic interaction chromatography column is butyl, octyl or phenyl based.

10. The process of claim 9, wherein the butyl based column is butyl Sepharose 4 fast (BS4).

11. The process of claim 9, wherein the octyl based column is octyl Sepharose 4 fast (OS4).

12. The process of claim 9, wherein the phenyl based column is Phenyl Sepharose (PSH, PS6-1 and PS6-h).

13. The process of claim 2, wherein the mixture containing retroviral particles and/ or soluble viral antigens is loaded onto the column in a solution having a salt concentration from about OJM to about 4M and a pH from about 6.0 to 8.5.

14. The process of claim 13, wherein the retroviral particles and/ or soluble viral antigens are eluted with a buffer at a pH of about 6.0 to 8.5.