CA2112226A1 - Purification of recombinant apolipoprotein e from bacteria - Google Patents

Abstract

Applicants describe methods for purifying human ApoE or analog thereof from recombinant bacterial cells with minimal protein aggregation and degradation during the purification process. The invention involves the addition of neutralized fatty acids to the medium during cell disruption and the use of a non-ionic detergent during the purification process. Additionally applicants describe a method for increasing the production of ApoE or analog thereof in a bacterial host by adding to the culture medium neutralized fatty acids, fatty acid precursors, triglycerides, triglyceride precursors or acetate. Pharmaceutical and diagnostic uses of the ApoE analog are described.

Description

~ 93/~3 21~ 2 2 ~ G PCTfUS91/~5~3 Purification of Recombin~nt Apolipoprotein E from Bacteri~.

Backaround of the,I~vention Apolipoprotein E (ApoE) is a protein found in the bloodstream in association with a variety of cholesterol and lipid-containing particles. The role o~ ApoE in metabolism has been reviewed by R.W. Mahley Science, 240: 622-630 (1988), and the complete amino acid sequ~nce of human ApoE
has been determined by Rall et al., J. Biol. Chem. 257(8):
417~-4178 (1982).

Through its ability to mediate lipoprotein binding and uptake by lipoprotein receptors, i.e. the LDL receptor and the chylomicron receptor, plasmatic ~poE (p-ApoE) has important functions in the regulation of plasma lipoprotein metabolism and in the maintenance of cholesterol homeostasis. ExtensiYe epidemiological studies have strongly indicated a correlation of high blood-cholesterol levels to heart attacks and strokes, due to the formatio~ of atherosclerotic plaques, the production of which is influenced by both genetic and environmental factors (M.S.
Brown and J.L. Goldstein, Science ~32: 34 (1986)).
Administration of exogenous ApoE ~ay assist in the regulation of serum lipid and cholesterol levels, and in the prevention of atherosclerosis.

In addition, ApoE is produced at a high level and accumulates in the region of injured and regenerating periph2ral nerves (M. J. Ignatius et al., Proc. Natl. ~cad.
,Sci. U.S.A. ~: 1125 (1986); G.J. Snipes et al., Proc. Natl.
Acad. Sci. U.S.A. 83: 1130; P.A. Dawson et al., J. Biol.
Chem. 261: 5681 ~1986)). ApoE may ba involved in the

2~ ~2 -2- PCT/US91~ 3 mobilization and possible reutilization of lipid or the repair, growth and maintenance of myelin and axonal membranes ~T. Vogel et al. ~oc. Natl. A~ad. Sci. U.S.A. 82:
8696 llg85)).
Apolipoproteins including ApoE have also been shown to have immunoregulatory activity tR.W. Ma~ley et al., J. Lipid Res.
25: 1277 (1984); R.W. Mahley & T.L. Innerarity, Bi_chçm-Biophys. Acta 137, 197 (1983)). ApoE-containing lipoproteins as well as low density lipoproteins have the capacity to inhibit or stimulate antigen-induced and mitogen-induced T lymphocyte activation and proliferation (R.W. Mahley, Science ~0: 62~-630 (1988)).

However, it i8 impossible to obtain from human blood sufficient quantities of naturally-occurring ApoE to examine the beneficial therapeutic effects which may be associated with ApoE. Accordingly, there is a need to provide a practical mean~ of producing suff~cient quantities of highly purified ApoE to conduct ani~sl and clinical trials and for widespread pharmac~utical use.

The subject inYention provid~s a novel purification ~thod for producing large guantities of highly purified, 2S biolcgically active, rQco~binant apolipoprotein E (r-~poE).
Thi~ method involves severa} novel features to solve inter problsms connected with ~l) aggregation and degradation of ApoE, (2) separation of active and inactive forms of ApoE, and (3) removal of endotoxins.
European Patent Publication EP 20S,715 assigned to Mitsubishi Chemical Industries Ltd. disclosed the cloning of ApoE in E. coli yeast ~nd CH0 cells. H~wever, the disclosure indicated that only minute amounts of ApoE were produced and did not include a purification method.

2112221i p~ 9 1 / 04 5 5 3 - -3-~3 R~ ? ~ Jl!N 1993 PCT Publication W0 8i 02061 assigned to Biotechnology Research Partners Ltd. disclosed the expression of fusion proteins containing the receptor recognition domain of ApoE
for drug deliver~. This publication does not disclose a method for production or purification of the full length ApoE analog protein.

PCT Publication W0 87 02059 also assigned to Biotechnology Research Partners Ltd. disclosed the correlation of polymorphisms in apolipoproteins (including ApoE) with atherosclerosis, but not a method for production or purification of ApoE.

Japanese Patent Application No. JP 61096998 assigned to Mitsubishi Chemical Industries Ltd. described the cloning and expression of apolipoprotein A-l analog.

Japanese Patent Application ~o. JP 60163824 assigned to Nippon Shinyaku KX described the use of serum lipoproteins including ApoE as drug carriers for intravenous injection.
The ApoE in JP 60163824 is not derived from recombinant sources.

Co-assigned, copending patent application~ U.S. Serial No.
896,750, filed on August 14, 1986 and U.S. Serial No.
08S,651, filed on August 14, 1987 (a CIP of U.S. Serial No.
896,750~ di~losed methods directed to small scale puri~ication of ApoE. While ~he recombinant ApoE analogs produced by these methods were very pure, it was necessary to develop a suitable method for industrial production scals-up. Further, it was desirable to prcduce an ApoE
analog containing lower endotoxin lavels than those previously produced. Specifically, the method disclosed in U.S. Serial No. 896,750 produces an ApoE analog which is greater than 90% pure but has an endotoxin level in the range of 500,000 - 1,000,000 pg/mg.

SUBSTITUTE SHEET

P~T;"~ 9 1 / 0 4 5 5 3 ~3 Rec'd P~ 2 8 J~N 1993 The method described i~ U.S. Serial No. 085,651 utilizes a urea solution throughout and involves batch chromatography on Phenyl- Sepharose, Heparin-Sepharose and DEAE-Sepharose columns. The resulting ApoE analog is greater than 95% pure and contains less than 2,000 pg endotoxin per mg. The use of urea necessitated the performance of the chromatography steps at 4C - 10C because urea is unstable and the ApoE
analog tends to degrade and aggregate in the presence of urea unless the experiments are performed in low temperatures.

Additionally, the chromatography methods in U~S. Serial No.
085,651 are batch methods involving overnight stirring, conditions which make scale-up very difficult. The endotoxin level of the resulting analog is still high.

The novel methods described herein, i.e. Scheme I and Scheme II, use Triton X-100 (TritonR) to protect ApoE from degradation and aggregation. TritonR allows the ultrafiltration and column chromatography step to be performed at room temperature. In addition, no batch steps are required in this process. The methods are therefore suitable for scale-up.
, J
Furthermore, Triton~ enables the use of acidic pH
condition~; thi~ is a distinct advantage ~ince acidic pH
conditions in the absence of Triton~ lead to precipitation o~ the ApoE analog. The addition of TritonR allows the enrichment of ApoE by an acidic pH extraction step and also the use of cation exchange chromatography.

The ApoE analog produced by the methods disclosed herein is extremely pure and has a very low endotoxin level, i.e.
about 25 pg/mg; therefore, the resulting ApoE analog can be used ~or animal trials. We have shown that the resulting ApoE analog can be lyophilized and still retains its S~.3Q~sT~TlJ~E SHEEr 2 ~ ~
W093/0~3 PCTJVS91/045~3 biological activity on sub~equent dissolutiQn, and we have demonstrated that the lyophilized ApoE is very stable.

W09 ~ , 6 ` PCT/US91/0~-~3 ~ri~f Dosor~tion o~ th- Fi~ur-~

F$gur- 1: Molecular Weig~t.of Recombinantand ~lasmatic ApoE
by Size Exclusion Chromatography The ApoE molecular weight, purity, and ~ggregation stat~ was determined by f~t perform~nce size exclu~ion c~romatography on a Superose 6 colu~n (HR 10/30, Pharmacia). A comparison of profiles of r-ApoE and p-ApoE is shown in this figure.
Panels A and B represQnt two different r-ApoE preparations, batches 01 and 02 respectively. These batches were produced from two different fermentations and were purified as described in Ex~mple 3. Panel C represents a p-ApoE
preparation and panel D repre~ents a buffer control. The retention time for both batches of r-ApoE and for p-ApoE
were 41.0, 41.37, and. 41.40 minutes, respectively both prepared as described in Example 3. The MW of both r-ApoE
and p-ApoE were estimated, from a MW standard calibration curve comprising 67 XD (BSA), 32 XD (superoxide dismutase) and 21 KD ~human growth hormone~, to be approximately 65 XD
indicating a dimer form. The additional peak of high MW
(about 5%) which appears in batch 01 (Panel A) may represent a slight aggregation in thiC preparation.

~igur~ 2: W ~çç~ra ~f Recombinan~ and Plasmatic ApoE

The W ~p~ctra of r-ApoE batch 01 (A) and batch 02 (B) were compared to p-ApoE. W absorbance was measured in the range of 350-200 nm, ~nd the spectra were found to be identical.
F~guI- 3: PFoduction of Plas~j~LpTVR 590-4 Plasmid pTVR 590-4, which thermoinducibly expresses met-ApoE
analog, is shown in this figure and is described in Example 1.

o 93/00443 ~ 1 1 2 2 ~ 6 Pcr/us9!/04s53 Figur- ~: Com~arison of Recombinant ~oE and Plasmatic ApoE
by SDS - Polvacrylamide Gel Electro~horesis Samples from each preparation were applied on to a 12.5%
-5 polyacrylamide-SDS gel. Lanes A and A' denote r-ApoE (Batch 01); lanes B and B' denote r-ApoE (Batch 02); lanes C and C' denote p ApoE. All lanes except for C' were loaded with approximately 25 ~g, while C' was loaded with 6 ~g. Lanes A, B and C were prepared with buffer containing 2-mercaptoethanol (2ME); lanes A', B' and C' were prepared without 2 ME. After electrophoresi~ the gel was stained with Coomassie blue reagent. The main bands for r-ApoE and p-ApoE migrate in a similar fashion. The low molecular weight (MW) band of r-ApoE (A, B) which appears just below the main band represents a cleavage product. The upper MW
band that appears in lanes A', B', and C' (material applied without 2ME) may represent sulfhydryl bonded multimers of ApoE. The p-ApoE (C) shows an additional band slightly above the main band which represents the glycosylated form of the plasmatic protein.

W093/~3 ~ PCT/US91/~3 ~u~ary of th- I~v~ntion:

The present invention provides method~ for purifying ApoE or analog thereof from ~ bacterial cell with minimal protein aggregation and degradation during the puri~ication process.
The preferred embodiment of the invention provides for a method of extracting ApoE or analog thereof from the cell pellet which comprises: (a) culturing bacterial cells which produce ApoE or analog; (b) disrupting the cell wall of the bacterial cell in a buffer containing magnesium ions to produce ~ lysate; (c) ~eparating cellular debris and insoluble precipitate from the ly~ate to obtain a pellet containing ApoE or analog; (d) ~olubilizing the separated pellet containing ApoE or analog with a solution containing a non-ionic detergent to obtain solubilized ApoE or analog;
(e) treating the solubilized ApoE or analog so as to concentrate and purify the ApoE or analog in the presence of a non-ionic detergent; and (f) recovering the resulting concentrated, purified ApoE or analog thereof.
The present invention also provides a method for purifying ApoE or an analog thereof from bacterial cell extract supernatant. Additionally the present invention pr,~vides for ~ method for increasing ~he production of ApoE or analog thereo~ in a bacterial host ~y adding to the culture medium neutralized fatty acids, fatty acid prçcursors, triglyceridQs, triglyceride precursors or acetate.

2 t 1222~
~093/0~3 PCT/US91/04553 _g_ , Dot~ D-s¢r~ptlon of th~ I~ve~t~o~

The present invention provides methods for obtaining a purified recombinant ApoE or polypeptide analog thereof from genetically engineered bacterial cells which produce the recombinant ApoE or polypeptide analog thereof. The preferred embodiment of the invention provides for a method of obtaining a purified recombinant ApoE or polypeptide analog thereof from genetically engineered bacterial cells which produce the recombinant ApoE or polypeptide analog thereof which co~prises: (a) culturin~ the genetically engineered bacterial cells so as to produce the recombinant ApoE or polypeptide analog thereof; (b) treating the bacterial cells in the presence of ma~nesium ions so as to obtain a lysate containing insoluble recombinant ApoE or polypeptide analog thereof; (c) recovering from the lysate insoluble material including insoluble recombinant ApoE or polypeptide analog thereof; (d~ treating the insoluble material 80 reco~ered with a solution containing a non-ionic detergent to obtain eolubilized recombinant ApoE or polypeptide analog thereof; (~) treating the solubilized recombinant ApoE or polypeptide analog thereof so~as to concentrate and purify the recombinant ApoE or polypeptide analog thereof; and (f) recovering the resultant concentrated purified recombinant ApoE or polypeptide analog thereof.

The invention i~ further exemp~ified as described below.
1. Bacterial ~ P~o~ucl2q ~oE A~aloq As used herein, an analog of ApoE is defined as a polypeptide having an amino acid seguence substantially identical to that of naturally occurring ApoE but differing from it by the addition, deletion or substitution of one or W093/~k~3 ~ ~ ~2~ o- PCT/US91/0 more amino acids while retaining the biological activity of naturally occurring ApoE>

The bacterial cell can be any bacterium in which a DNA
sequence encoding apolipoprotein E or an analog thereof has been introduced by recombinant DNA technique~. The bacteria must be capable of expres~ing the DNA sequence and producing the protein product.

The bacteria can be any ~train including auxotrophic, prototrophic and lytic strains, ~ and F strains, strains harboring the cI~7 repressor sequence of the lamkda prophage and strains wherein the deo repressors or the de~ gene have been deleted.
Examples of wild type E. coli strains are prototroph ATCC
No. 12435 and auxotroph MCl061 (ATCC No. 67361).

Examples of ~. coli strains which harbor the lambda cI~7 repressor sequence are ~he auxotrophs Al645 containing plas~id pTVR 279-8 ~ATCC No. 53216~, Al637 containing plasmid pT~lO4(2) (~TCC No. 39384) and ~2097 containing plasmid pSOD~2 (ATCC No. 39786); ~nd the prototrophs ~4200 containing pla~id pHG44 ~ATCC No. 53218) and biotîn-independent A4346-containing plasmid pHG44 (ATCC No.
53218).

An exa~ple of a lytic E. cQli strain is A4048 containing plasmid pHG44 (ATCC No. 53217~.
Examples of F strains are Sp930 (F) containing placmid pMF
5534 deposited under ATCC No. 67703 and E coli W31100 (F-) containing plasmid pEFF 920 deposited under ~TCC No. 67706.

Examples of E. coli strains where.in the deo gene or deo repressors have been deleted are Sp732 which contains ` WO 93/00443~ ` . 2 1 i 2 2 2 6 PCI`/US91/04~3 plasmid pMF 2005 (ATCC No. 67362), Sp540 which contains plasmid pJBF 5401 (ATCC No. 67359) and Sp930 which contains plasmid pEFF 920 (ATCC No. 67706).

All the E. coli host strains described above can be "cured"
of the plasmids they h~rbor by methods well-known in the art, e.g. the ethidium bromide method described by R.P.
Novick in ~ ç~iol. Review 33, 210 (1969).

The bacterial cell may contain the DNA sequence encoding the ApoE or analog in the body of a vector DNA molecule such as a plasmid. The vector or pl~mid is constructed by recombinant DNA techniques so that the sequence ~ncoding the ApoE is incorporated at a suitable position in the molecule.
Plasmids which are used for ApoE analog production can harbor a variety of promoters such as the lambda promoter or the deo promoters.

Among the plasmids which may be used for the production of the ApoE analog are as fol~ow~:

A. Pla~mid pTVR 590O4 deposited in . oli 12435 (ATC~ No.
67360~, B. Pla~mid pTVR 279-8 deposited in E~ coli A1645 (ATCC No.
~32~

C. Any pla~id derived from the above plasmids or from the plasmid pApoE-Ex2 deposited in Eo coli A1645 ~ATCC No.
3g7~7)-D. Any other plasmid which expresses ApoE.

35 2. F-r~e~tat~o~ o~ ¢~ xPro~ ~ apo~ ~n~lo~

2~2226 PCT'U~ 91/045 5 3 r ` 03 Re~ PM/,~ J~ 1993 --12 ~

we found that bacterial cells expressing the ApoE analoq are subject to a "toxic effect" which causes cell lysis soon after ApoE induction. We discovered that protection of the host bacterial cell from this "toxic effect" was achieved by adding to the culture medium an inhibitor of proteolytic digestion of ApoE or analog thereof such as neutralized fatty acids, triglycerides or triglyceride precursors, including any of (i) the fatty acids, e.g. sodium propionate, n-butyric acid (neutralized), and beta-hydroxybutyric acid (neutralized); (ii) the triglycerides,e.g. triacetin, tributyrin and tricaprylin; (iii~ the triglyceride precursors, e.g. 1-monomyristoyl-rac-glycerol, 1-monopalmitoyl-rac-glycerol, DL-alpha-hydroxy isovaleric acid and glycerol and (iv) the fatty acid precursor sodium acetate. Butyric acid, beta-hydroxybutyric acid, propionic acid and sodium acetate are preferred and sodium acetate is especially preferred.

The protection of the host cell as described above enabled applicants to employ a long (3 hours) induction period at 42C instead of the short (15 minutes) induction period previously disclosed (see coassigned copending U.S. Serial No. 085,651), and facilitated the scaling up of the fermentation process for industrial production.
Whil~ not wishing to be bound by theory we present the foll~wing rationale to explain these results.
Apo~ipoproteins are known to interact with lipids and lipid-containing moieties. The "toxic effect" may be explained by the interaction of ApoE with the bacterial cell membrane or~by binding of precursors which are important for membrane biosynthesis. If this reasoning is correct the host E. coli cell is protected by adding to the medium compounds such as fatty acids, fatty acid precursors, triglycerideR or glyceride precursors, with the assumption that théy penetrate the cell, bind to ApoE or to other SU~3STITUTE SHEET

"W093/~3 2 ~ :; 2 2 2 ~ PCT/USgl/045~3 cellular components and thus prevent the ApoE toxicity.

We also found that the aforesaid compounds and also EDTA
protect the ApoE protein from proteolytic degradation.

3. C~ll Di~rupt~o~

Surpri~ingly, we found that adding fatty acids to the medium protected the ApoE analog from degradation by cellular proteases. This was confirmed by in vitro incubation studies, as described in Exa~ple 7 and it was therefore decided to add ~uch substances to the buffer during disruption of the cells. Beta- hydroxybutyric acid, butyric acid or n-caproic acid (all neutralized~ are preferred as inhibitors of proteolytic degradation of ApoE analog; for reasons of cost and con~enience, beta-hydroxybutyrate (sodium salt) is especially preferred.

E. coli cells containing r~combinant ApoE analog are harvested and disruptad in a buffer containing bivalent cation preferably magnesium ion (19-100 ~M) and an inhibitor of proteolytic degradation of ApoE such as beta-hydroxybutyrate at concentra~ions of 0.$-1%. Any cell disruption method can be used, e.g. sonication, mechanical disruption such as glass bead grinding (~ynomill), or explosion by pres~ure (Gaulin homogenizer). Under these condition~ ApoE analog above is precipitatad with the cell debris and i~ separated from ~o~t solu~le cytoplasmic componente by centrifugation. (See Scheme I and Example 3.) An alternative aspect of the invention describes production of ApoE analog from the supernatant. The cells are disrupted as described above except that the buffer does not contain magnesium ions; instead the buffer contains a chelating agent such as EDTA.

W093/~3 ~ 6 -14- PCT/US91/0

4. ~tractlon o~ ApO~

The pellet containing the ApoE analog is suspended in a buffer containing non-ionic detergent. Preferably the non-ionic detergent is EmulphogenR-BC720 (Sig~a) or PEG
(9-10) p-t- octylphenol which is sold under the tradename TritonR X-lO0 (Merck), designated TritonR. TritonR was used at a concentration of 0.05-~ preferably 0.3%, at pH between 3.o and 9.O, preferably 3.0 to 5Ø Under these conditions ApoE analog i8 selec~ively extracted from the pellet and is protected from degradation and from formation of aggregates.
The extract is neutralized to pH 7.5 and ultrafiltrated.

An optional step before ultrafiltration is to filter the supernatant solution to r~move turbidity. This is done particularly with large sa~ples (15 K~ bacterial cake). The preferred filter is the 0.4-l.0 micron depth filter zeta plus 50 SP of Cuno Inc., Industrial Filter Products, 400 Research Parkway, Meriden, CT 06450, U.S.A.

5. ~ltra~ltr~tio~

Any appsopriat~ ultrafiltration ~ethod with a cutoff ~oint of about lO0 K which d~ not allow ~poE analo~ to pass5 through the filter ~y be used for the fractionation of the ApoE ~nalog. Ex~mple~ of ~;uch methods are 2~illipore ' s Pellicon c~ssette system (the preferred system) or Amiconls hollow ~iber system. In all following purif ication steps there is the novel addition of non-ion~c deterg~nt, e . g .0 EmulphogenR or TritonR, preferably TritonR, at concentrations of 0.05-1%. In the presence of TritonR, ApoE does not cross the memb~ane; it is s~p~rated from low molecular weight components and is dialyzed against the equilibration buffer of the next purif ication ~tep.5

6. Colu~ c~ro~to~ra~hy n-thQ~

W093/~J3 2 1 1 2 2 2 ~ PCT/US91/045S3 A) DE~E Se~harose Any weak anion exchange chromatography method can be used in this step but DEAE-Sepharose fast flow (Pharmacia) is preferred. Weak anion exchange columns usually have as functional group a tertiary amine tdiaminoethyl), but amino ethyl is also possible. The matrix may be based on inorganic compounds, synthetic resins, polysaccharides, or organic polymers; possible matrices are agarose, cellulose, trisacryl, dextran, glass bead~, oxirane acrylic beads, acrylamide, agaro~e/polyacrylamide copolymer (Ultrogel) or hydrophilic vinyl polymer (Fractogel).

The retentate solution from the previous step is chromatographed on a DEAE Sepharose column in buffer, preferably Tris buffer, at a range of pH 7-8, preferably at pH 7.5. ApoE analog i5 eluted from the DEAE column with salt solution preferably sodium chloride.

The ApoE fraction eluted from the DEAE column has the same molecular weight as ApoE from human plasma when analyzed under the sa~e conditions. This fraction is dialyzed and equilibrated w~th ~he buffer u~d for loading the~ next column by ultrafiltratio~ dialysi~ using a 100 K membrane as 2S described above in section 4.

B) Qt~3~1L2~

Any strong anion exchange (e.g. guaternary amine) method can be used in this step but Q-Sepharose (Pharmacia) chromatography is preferred. The functional group of the ion exchange column can be any quaternary amino group such as quaternary amino ethyl and the matrix can be any of the matrices listed hereinabove at paragraph (A)J The dialyzed sample from the previous step is loaded on a Q-Sepharose column in Tris buffer containing TritonR at a concentration ~} ~?~ 16- PCT/US91/~ '~

in a range of 0.1-0.5%, preferably at 0.2% TritonR. At low salt concentrations (up to loO mM NaCl) an active ApoE
fraction containing low levels of endotoxin~ that has the same pI as plasma ApoE is eluted and is separated fro~ an lnactive fraction of ApoE that i~ eluted at higher salt concentrations (above 200 mM NaCl).

The eluted actiYe fraction of ApoE i8 dialyzed against the equilibration buffer used for loading the next column by ultrafiltration dialysis using a lOOK membrane as described above in section 4.

C) CM-Sepharose Any cation exchange (e.g. c~rboxymethyl) me~hod can be used in this step but CM-Sepharose fast flow (Pharmacia) chromatography is preferred. The functional group can be carboxymethyl, a pho~pho group or sulphonic groups such as sulphopropyl. Po#sible matri~e~ are list~d her~inabove at paragraph (A). The dialyzed sa~ple i8 chromatographed on C~-Sepharose colu~n in sodium acetate buffer pH 4.0-6.0, preferably pH 4O8-5~2~ containing O . 2% Triton~ and then washed and eluted w~th ~uff~r containing 0.05% Tr~onR.
This ~tep enables r~mo~al of endotoxins from Apo~ requlting in very low endotoxin levels, less than 100 pg endotoxin per mg ~poE analog and usually betw~en abou~ 20 to about 50 pg per mg. In addition, this step r~duces the concentration of Triton~ to 0.05%.

7. Tr~to~~ Ro~al TritonR is removed from the above sample by ultrafiltration dialysis using a 100 X membrane. Any ultrafiltration method with a cut off point of 100 X may be used as described above. Dialysis is carried out against buffers without TritonR at ApoE concentrations of 0.5 ~g/ml to 2 mg/ml and ~ W093/0~3 2 3 1 2 2 2 6 PCT/US91/04~3 TritonR concentrations below its critical micelle concentration.

There are three conditions which must prevail during the Triton~ removal step. These are detailed in Example 3G.
These conditions allow fast removal of TritonR while retaining the ApoE in non-aggregated form. The ApoE analog produced is biologically active and very pure. The ApoE
analog can then be lyophilized and regain full ~iological activity on subsequent redissolution.

In a preferred embodiment of step (a) of the above described method the bacterial cell is Escherichia coli but other bacterial cells could be used. Further, in the same preferred embodiment, in step (b) the ce~l wall is disrupted mechanically. Moreover, the separation of cellular debris and insoluble precipitate from the lysate in step (c) comprises centrifugation but other separating ~e~hods could be used. Additionally, the non-ionic detergent utilized in the above-described preferred embodiment is TritonR but other non-ionic detergents could be used.

In a preferred e~bo~iment of step (d) of the above-described method, the treat~ent ætep utilized to concentrate and purify the ApoE or analog comprises ultrafiltration, but thi could ~e replaced or complemented by other concentration and filtering methods such as amonium sulfate precipitation, dialysis, freeze- drying or centrifugation.
Moreover, the ultrafiltration removes components of molecular weight less than 1 x 105 daltons. Further, he preferred embodi~ent of treatment in step (e) comprises chromatography, with the ~ost preferred being ion exchange chromatography. The most preferred embodiment comprises ion exchange chromatography comprising a tertiary amine ligand attached to a resin. After the chromatography step the resulting concentrated, purified ApoE or analog is dialyzed~

W093/~3 PCT/US91/~ -~
~ 18--saving the retentate. The retentate containing the resulting concentrated, purifi~d ApoE or analog are further concentrated and purified by using another chro~atography step preferably chromatography co~pri~ing a guaternary amine ligand attached to a resin~ Following the chromatography step the resulting concentrated, purified ApoE or analog is dialyzed, ~aving the retentate.

The retentate containing the resulting concentrated, purified ApoE or analog is further concentrated and purified by ion exchange chromatography comprising a carboxy methyl-ligand attached to a resin. The recovery of the concentrated, purified ApoE or analog in step (f) of the preferred method comprises ultrafiltration. The ultrafiltration remove~ molecule~ of molecular weight less than 1 x 105 daltons.

W093/0~3 2 ~ 3. ~ PCT/US91/04SS3

8. A~LAl~-r~at~- ~mbo~ nt of t~ vont~o~: Purif~c~t~on o~ Aoo~ Analog from t~ ~uD-rnatant The present invention additionally provides another method s for obtaining a purified recombinant ApoE or polypeptide analog thereof from genetically engineered bacterial cells which produce the recombinant ApoE or polypeptide ana}og thereof which compri es: (a) culturing the genetically engineered bacterial cells so as to produce the recombinant 10 ApoE or polypeptide analog thereof; (b) treating the bacterial cells in the presence of EDTA so as to obtain a lysate containing soluble recom~inant ApoE or polypeptide analog thereof; (c) recovering from the lysate a solution containing soluble recombinant ApoE or polypeptide analog 15 thereof; (d) treating the solution so recovered with a solution containing a non-ionic detergent to obtain a second solution containing solubilized recombinant ApoE or polypeptide analog thereof; (e) treating the second solution containing recombinant ApoE or polypeptide analog thereof so 20 as to concentrate and purify the recombinant ApoE or polypeptide analog thereof; and ~f) recovering the resultant concentrated purified recombinant ApoE or polypeptide analog thereof.

25 The extraction of ApoE analog from the supernatant is similar to that described for extraction from the pellet but the following points must be noted:

i) Extraction with Triton~ is similar except the preferred 30 pH is 4.0-4.5.

ii) Ultrafiltration is as described hereinabove and serves the same function.

Og3/0~3 PCT~US9l/~'-~o-iii) DEAE SeDharose ChromatoaraDhY

DEAE Sepharose (weak anion) chromatography separates theApoE analog into two fractions. At low salt concentrations (up to about 80 mM NaCl) an active ApoE fraction containing low levels of endotoxins that have the same pI as plasma ApoE is eluted and is separated from ~n inactive fraction of ApoE analog that contains a high endotoxin level that is eluted at high salt concentration tabove 150 mM NaCl).
Thus, the weak anion DEAE- chromatography step functions in Scheme II as the Q-Sepharose step in Scheme I.

d) Heparin-Se~harose Chromatoara~hv Any affinity exchange chromatography method with a functional group that binds ApoE ca~ be used in this step, e.g. dextran sulphate or chondroitin sulphate, but heparin is preferred. The matrix used can be any of the matrices listed above in section 6 for use in purifying ApoE from the pellet. However, Sepharose i8 th~ preferred matrix. The buffer containing Triton~ can bQ in the pH range 6.5 to 9.5 but pH of about 7.0 i~ preferred a~ giving a higher loading capacity of ApoE analog. Thi~ step remove~ ç~i pr~tein impuritie~ and endotoxin~ re~ulting in very low levels of endotoxin (about 60 pg~g).

iV) ~ S~R >~ t These steps have the ~ame purpo~e and operate under similar conditions as in the preferred e~bodi~ent for purification of ApoE from the pellet.

In a preferred e~bodiment of the above-described method for purifying ApoE or an analog thereof from bacterial cell supernatant the bacterial cell in step (a) is preferably Escherichia coli but other bacteria may be used. The cell wall in step (b) is disrupted e~g. mechanically. Further, the separation of cellular debris and insoluble precipitate from the cell supernatant in step (c) comprises centrifugation but other methods of separating may be used.
s Additionally, the non-ionic detergent utilized throughout the method for purifying ApoE or an analog thereof from bacterial cell supernatant is preferably TritonR;
alternatively, the non-ionic detergent is EmulphogenR.
Further, the treatment in step (d) to concentrate and purify ApoE or analog comprises preferably ultrafiltration but other concentration and filtering methods could be used.
The ultrafiltration removes molecules of molecular weight `~-less than 1 x 105 daltons.

Further, the preferred embodiment of treatment in step (e) ;~
comprises chromatography with the most preferred comprising ion exchange chromatography. The most preferred ion exchanger is a tertiary amine ligand attached to a resin.
Following the chromatography step the resulting concentrated, purified ApoE or analog is dialyzed, saving the retentate. The retentate containing the resulting concentrated, purified ApoE or analog is further concentrated and purified preferably by affinity exdhange chromatography wherein the most preferr~d a~finity exchanger is a heparin ligand attached to a resin. The resulting concentrated, purified ApoE or analog is dialyzed, again sa~ing the retentate. The retentate containing the resulting more concentrated, purified ApoE or analog is further concentrated and purified preferably by cation exchange c~romatography wherein the most preferred cation exchanger is a carboxymethyl ligand attached to a resin.
Further, the recovery of the concentrated, purified ApoE or analog in step (f) preferably comprises ultrafiltration which removes molecules of molecular weight less than 1 x 105 daltons ~ut other methods of concentration and filtering could be used.

2 ~2 ~ -22- PCT/US91/~4~

Additionally, the invention provides a CompoSition comprising ApoE or analog thereof produced by the above-described methods and a ~uitable carrier and also provides a co~position comprising such ApoE which contains or is linked physically to a chemotherapeutic or radiotherapeutic or radiodiagnostic agent and a suitable carrier.

Further, the pre~ent invention provides a method of treating a subject suffering from atherosclero~is which comprises administering to the ~ubject an amount of ~poE or analog thereof effective to combat atherosclerosis. As used herein atherosclerosis means a variable combination of changes in the intima of arteries consisting of the focal accumulation of lipids, complex carbohydrates, blood and blood products, fibrous tissue, and calcium deposits, and associated with medial changes.
t Additionally, the invention prov~des a method of treating a subject suffering from hypercholesterolemia caused by impaired chole~terol metabolism which comprises administering to the ~ubj~ct an amount of ApoE or analog thereof effective to normalize cholesterol meta~olism so as to alleviate hyparchole~terolemia and thereby trea~ ~he subject. As us~d herein hyperchole~terolemia means an 2~ excesg of cholesterol in the blood.

Th~ presQnt invention further provides a method of treating a subject suffering from hyperlipoproteinemia caused by impaired lipid metaboli~m which comprises administering to the subject an amo~nt of ApoE or anslog thereof effective to normalize lipid met~bolism so as to alleviate hyperlipoproteinemia and thereby treating the subject. As used herein hyperlipoproteinemia means an excess of lipoproteins in the blood.
Additionally, the invention provide~ a method of treating a ~W093/~3 ~ S PCT/US91/04553 subject suffering from neuronal injury which comprises administering to the subject an amount of ApoE or analog thereof effective to pro~ote nerve development and regeneration.
:
Furthermore the invention provide~ a method of trezting a ~:
subject suffering from a tumor which harbors high levels of LDL rQceptor which comprises admini6tering to the subject an amount of ApoE or analog thereof which contains or is linked physically to a chemotherapeutic or radiotherapeutic agent effective to treat the tumor.

Additionally the invention provides a method of diagnosis of LDL receptor defects in a subject by ad~inistering to the subject an amount of ~poE or analog thereof which contains or i8 linked physically to a radiodiagnostic agent effective to quantitate the LDL receptors.

Furthermore the invention provides a method of diagnosis of primary or secondary sites of tumor growth in a subject where the tumor harbors high level~ of LDL receptors which comprises administering to the subject an amount of ApoE or analog ther~of which contains or is linked physicall~ to a radiodi~no~tic agent effective to visualize the tumor.
Furthermore the invention provides a method of treatment of autsimmune di~ease in a subject which comprises administering to the subject an amount of ApoE or analog thereof effective to treat the subject.
Furthermore the invention provides a method of treatment of a subject having an immunodeficient disease which comprises administering to the subject an amount of ApoE or analog thereof effective to treat the subject.
In addition the invention provides a lipid emulsion PCT/U~ 91 / 0~5 ~ 3 21 i 2 2 2 ~4 03 Re~'fJ P~lIP~ ~ 8 ~U~ 1993 .
comprising ApoE or analog thereof wherein the ApoE or analog thereof is a ligand, and the use of such lipid emulsion for drug delivery and tissue targeting.

The invention further provides a method for increasing the production of ApoE or analogs thereof in a bacterial host by adding to a medium in which the host i~ growing an effective amount of compounds selected from the group consisting of neutralized fatty acids, fatty acid precursors, triglycerides, triglyceride precursors and acetate. In a preferred embodiment of the invention the neutralized fatty acid is sodium propionate, n-butyric acid, or beta-hydroxybutyric acid. In another preferred embodiment of the invention the triglyceride is triacetin, tributyrin or tricaprylin. In a further preferred embodiment of the invention the triglyceride precursor is 1-monomyristoyl-rac-glycerol, 1-monopalmitoyl-rac-glycerol, DL -~-hydroxy isovaleric acid or glycerol. In a most preferred embodiment of the invention the added compound is acetate, preferably sodium acetate at a concentration of about 0.1% - 1$ and the preferred concentration is about 0.5%. The preferred concentration in the culture of fatty acid, triglyceride or glyceride precursor is about 0.1% - 0.5%, and the preferred concentration is about 0.2%. 4 Finally, the invention provides a method of prot2cting ApoE
protein in solution from degradation by adding to the solution an effective amount of a compound selected from the group con~isting of neutralized fatty acids, fatty acid precursors, triglycerides, triglyceride precursors, acetate and EDTA. In one embodiment of the invention, the effactive amount of compound added to the solution produces a concentration of compound in the solution of about 0.1% to 0.5%, preferably about 0.2%.
The methods of the present invention will be better S~S~lTUTE SHEE~

~WO93/0~3 2~ 6 PCT/US91/04553 understood by reference to the following experiments and examples, which are provided for purpo~es of illustration and are not to be con~trued as in any way limiting the scope of the invention, which i~ defined by the claims appended.

W093/0 ~ 3 ~c~ 26- PCT/US91/04~'~

ES~MPLE 1 Host-Vector System For ADoE Analoq Exorçssion The preferred host-vector sy~tem used for production of the ApoE analog met-ApoE i5 E~ coli strain ATCC No. 12435 harboring plasmid pTVR 590-4; this ho~t-vector system, shown in Figure 3, has been deposited with the American Type Culture Collection (ATCC) in Rockville, Maryland under ATCC
Accession No. 67360.

The construction of plasmid pTVR 590-4 which i~ described below has been fully described in co-assigned copending patent application, EPO Publication No. 303,972 (Exa~ple ll and Figure 27). Plasmid pTVR S90-4 contains the following elements: .

a) Origin of replication.

b~ The Amp~ gene in counter clockwise orientation.

c) In clockwi~e orientation and in 5' to 3' order, a truncated qeo Pl pro~oter sequence and the l~mbda cI~7 te~perature-sensitive repre~sor roding sequence.

d) In counterclockwi~e orientation and in S' to 3' order, the lambda pro~o~er, the beta lactamase pro~oter-ribosomal binding site, the coding sequence for ApoE analog and the T~T2 transcription ter~ination sequences.

This plasmid is a high level expressor of ApoE analog protein under the control of the strong leftward promoter of bacteriophage lamkda (P~) which is thermoinducibly controlled by the constitutively expressed cI857 ~~093/~3 2 1 1 ~ 2 2 ~ PCTIUS9l/04553 temperature-sensitive repressor also situated on the plasmid. Production of ApoE analog protein from this plasmid takes place only on heat- induction at 42OC.

This is a so-called "ho~t independent" expression system since the plasmid can thermoinducibly produce the met-ApoE
analog independent of prior insertion of the lambda cI857 gene into the host E. coli chromosome. This plasmid can therefore be used to transform a wide variety of host bacterial cells. The host described, ATCC No. 12435, is a prototrophic wild-type strain of ~. coli freely obtainable from the ATCC collection.

WO 93/00443 ?,~ 6 PC~/US91/04~C3 i 2 Growth of E. coli ATCC Accession No. 12435 harborinq plasmid DTVR 590-4 and ~roduction of bacterial cake containina A~oE
analoa The following description of the fermentation of E. coli producing ApoE analog is the preferred embodiment for production of cell cake containing ApoE analog.
1. Seed Flask Developmen~

The contents of frozen vials containing E. coli ATCC No.
12435/pTVR 590-4 (Example 1) are u~ed to inoculate seed flasks containing the following ~edium:

K2HPO4 9 g KH2PO4 1 g NaCl 5 g MgSO4.7H20 0.2 g NH4Cl 1 g FeNH4 citrate 0.01 g Trace ele~ent~ ~olution 1 ml ,~
Biotin 0.5 mg Glucose 5 g Ampicillin, sodium salt 0.1 g ~e~onized water 1 L

Trace elemen~s stock solution:
MnSO4~0 1 g ZnS04 .7H~0 2.78 g CoCl2.6H20 2 g Na~MoO4~2H2 2 g CaCl2 .2H20 3 g CuSO4 5~2 1.85 g H~BO~ o.s g ~W093/0~3 2~ ~?~ PCT/US91/045~3 Concentrated HCl 100 mL
Deionized Water 900 mL

Glucose and ampicillin are added from sterile concentrated stock solutions after autoclaving the other components of the ~edium. The cultures are incub~ted at 30C overnight on a rotary shaker at 250 rpm, and reach an O.Du~ of 3.5-5Ø

2. Seed Fer~e~er The contents of the seed flask are used to inocul.ate a 50 L
seed fer~enter containing 2S-30 L of the following production medium, which contains per liter:
K2HP04 8 g KH2~04 2 g Sodium citrate 2 g NH~Cl 2 g FeN~ citrate 0.02 g CaCl2.2H20 0~04 g 2 0 K2S04 0 . 6 g Trace ~lement~ solution 3 mL
(as in Section 1) Antifoam (Silicolap~e 5400) 2 ~L
2~

W093/00~3 ~ ~ PCT/US9l/0~ ~3 Added a~ter steriliz~tion (per liter of medium) MgS04 .7~0 . 0.4 g Sodium ampicillin 0.1 g Glucose 40-60 g NH~ (25-28% in water) approx. 40 mL

Glucose i8 added batchwise at inoculatio~; am~onia is automatically added for pH control (set point pH=7.0) during growth.

The culture is cultivat~d at 30C ~or 15-20 hours in order to achieve growth; the O.D~ gener211y reaches 20-30 during this time. This is equivalent to a dry cell weight (DCW) of 7.5-12 g/L.

3. Production Fermenter The contents of the ~eed fermenter are used to inoculate a 750 L ~nominal volume) fer~enter containing about 360 L
production ~edium as d~cribed for seed fer~entor, but excluding ampicillin. The culture is cultivated at 30C
until an O.D~ of lO iæ obtained. Induction of ApoE ~nalog expres~ion i8 then achiev~d ~y raising the ~ermenter temperatur~ to 42C. At induction, the following arg added to the fermenter:

DL-methionine 0.6 g per L of medium Sodium acetate 5 g per L of ~edium The sodium acetate (0.1% - 1%) i~ added to protec~ cells from the "toxic e~fect" caused by the ApoE analog.

The fermenter temperature is maintained at 42C for three hours, at which time the cells are harvested. The O.D~o of the cell suspension at harYest is generally 16-20, the ~W093/0~3 2~ 1~2~6 PCT/US91/04553 volume is 400-430 L and the D~W is 5.0-6.5 g/L.

4. Ha~ç~_f ~Çll~

The cell suspension is centrifuged at 14,000 rpm (16,000 g) in a CEPA lOl tubular bowl centr~fuge ~t a feed rate of 250L/hr, and a cell cak~ weighing about lO Kg is produced and saved. Alternatively, the cell suspension is centrifuged in a Westfalia CSA-l9 continuous centrifuge at 500 L/hr. The sludge is either disrupted immediately or frozen.

In both ca~es the supernatant contains no detectable ApoE
analog as measured by SDS-polyacrylamide gel electrophoresi~.

W093/0~3 PCT/US91/O~Fi3 ~A~ 32-gXl~SPLI~ 3 r Improved Method for Purification of Recombinant ApoE Analoq S The following ~ethod is suitable for ~cale-up for industrial application and yields ~ very pure ApoE analsg~ The general scheme of the downstrea~ process (Scheme I) consists of steps A through G as follows:

Scheme I. Downstream Proce~sina of the Recombinant Human A~oE Analoa Extracted fr~m thç Pellet IONS.
B EXTRACTION OF CELL PELLE~ WITH TRITONQ.

C lOOK ULTRAFILTRATION.

D DEAE CHROMATOGRAPHY

E Q SEPHAROSE CHRONATOGRAPHY
, --F CM SEPHAROSE CHR~M~TOGRAPHY
G lOOK ULTRAFILTRATION - ~RITONP REMOVAL.

The following detailed example sf the steps in purification of the ApoE analog is an example involving 3 Rg cell cake.
In addition we have successfully processed a 15 Kg cell cake using the methods described below with only minor modifications involving scale-up in the size of the equipment used.

Steps A through D were performed on 2 batches of bacterial cake, each weighing l.S Kg. After step ~, the two batches ^~WO93/~k~3 2 ~3~ 2 6 PCT/US91/04553 were combined and processed as one batch through steps E to G. Steps A, B, C were performed at 4C - 10C, except where otherwise indicated. All other activities were performed at room temperature.
s A. CELL DISRUPTION IN PRESENCE OF MAGNESIUM IONS

l.5 Kg of wet cell cake was suspended in 6 L of buffer A
which consist~ of 50 mM tris/HCl, 30 mM MgC12, 0.25% beta hydroxybutyrate sodium salt, pH-7.5. (The beta hydroxybutyrate wa~ added a~ a protea~e inhibitor; see Example 7.~ Thi~ w~s then homogenized using a Kinematica homogenizer yielding 7.5 L of homogenate. Disruption was then performed using a Dynomill XDL bead mill disrupter (Willy A. Bachofon, Ba~el) at 5 L/hr ~in two cycles).
Three-fold dilution of the resulting suspension using buffer A yielded a volume of 22.5 L. This lysate contained about 6 g ApoE analog, i.e. about 4 g ApoE analog per Kg of original bacterial cake.
Centrifugation wa~ then perfor~ed in a continuous CEPA-41 tubular bowl centrifuge, (Carl Padberg, Lahr/Schwarzwald) with a feed rate of 9 L/hr at 20,000 rpm (l7,000 g).' The pellet, weighing approximately 700 g and containing the insoluble ApoE analog wa~ saved and the supernatant was discard~d. (Note ~hat the ApoE is in~oluble due to the presence of Mg~ ion~.) B. EXTRACrION OF CELL PELLET WITH TRITO~
Six liters (l:lO) of extraction buffer were added to the pellet. (Extraction buffer: 50 mM tris/HCl, 20 mM EDTA, 0.3% TritonR, pH adjusted to 3.0 with HCl). Suspension was achieved using a homogenizer (Xinematica) at low speed.
Then another 6 L extraction buffer was added (giving a final pellet:buffer ratio of 1:20) and the pH was adjusted to 4.5 W093/0~ PCT/US~1/04'~3 with 1 N NaOH. The re~ulting 12 L ~u~pen~ion was incubated for 10 minutes at room temperature with stirring.

After incubation, the suspension was centrifuged on the CEPA
41 Centrifuge at a feed rate of 20 L/hr. The pellet weighing about 450 g wa& discarded and the supernatant solution containing ApoE analog was titrated to pH=7.5 with 1 N NaOH and saved.

Note: TritonR is present in all following steps and is removed in step G. ~;

C. 100 X ULTRAFILTRATION

The purpose of this step i8 to remove low molecular weight contaminants by ultrafiltration/dialysis.

A M~llipore Pellicon ultra~iltration ~ystem using one i00 K
cassette type PTHK wa~ utilized to concentrate the ;
supern~tant of the previous step (about 12 L) to about 2 L.
The feed pressure was 20 p5ig and the filtrate flow rate was 20 L/hr. The dialysis buffer was 50 mM tris HCl, 10 mM EDTA
and 0.1% Tr~ton~, pH-7.5. T~e 2 L retentate cont~lning about 2 3 mg ApoE analogJ~l was kept cool with ice~
The retent~te was dialyzed using the recirculating mode of the Pellicon ultrafiltration system until a filtrate conductivity equivalent to that of the dialy~is buffer was obtained; thi~ was the criterion used throughout the purific?tion for termination of dialysis.

D. DEAE CHROMA~OGRAPHY

The purpose of this step is to ~eparate th~ ApoE analog from contaminants such as proteins and other cellular materials.

_~093/~3 2 1 ~ 2 2 ~ ~ PCT/US91/~553 In this step a 1.6 L DEAE Sepharose Fast Flow column (Pharmacia) was used. The flow rate was 10 column volumes/hr. The capacity of the column under these conditions was determined to be 4 mg ApoE/ml. The column s was first equilibrated with DEAE equilibration buffer: 20 mM
tris/HCl, 1 mM EDTA, 0.5% TritonR, pH-7.S.

The retentate ~olution from the previou~ step (about 3 L) was then loaded on the column and washed with 3 column volumes (CV) of equilibration buffer. The first elution was performed using 3 CV of equilibration buffer containing 120 mM NaCl. Fractions were collected and the progress of the run was monitored by continuou~ly following the absorbance of the eluate at 280 nm. The fractions were analyzed by SDS
polyacrylaJide gel electrophoresi~ ~tained by Coomassie Blue and the trailing edge of the peak (3.1 CV) was saved.

The second elution was performed using the eguilibration buffer containing 150 mM NaCl. Fractions were collected and analyzed by SDS gel electrophoresis and most of the peak (3.9 CV) wa~ sav~d. Endotoxins were measured by the Limulus Amebocyte Ly~ate (LAL~ assay descr~bed in U.S~ Pharmacopeia (U.S.P.) XXI, 1165-1166 S1985). ~he l~vel of endotox~ was 3 ~g per mg ApoE analog.
Concen~ration and dialysis a~t~r DEAE~$e~arose Th~ fractions indicated fr~m the first and second eluates were pooled and dialyzed using the Pellicon ul~rafiltration system, with one lOOX ca~sette; the ~ialy~is buffer was 20 mM tris/HCl, 1 mM EDTA, 0.1% TritonR, pH=7.5. The sample was concentrated to 2 L (about 2-3 ~g ApoE/ml) and dialyzed.

E.

W093/~3 PCT/US91/04rC3 ~ 36-The purpose of this step is to separate the active from the inactive ApoE analog and to further remove endotoxins.

In this step a 1.6 L QS Fast Flow Column (Pharmacia) was used; the column capacity under the~e condition~ was about 7 mg ApoE/ml and the flow rate was about 10 CVJhrr The QS equilibration buffer wa~ 20 mM tris/HCl, 1 mM EDTA, 0.2~ Triton~, pH-7.8. After equilibration, the retentate solutions from two batches of the previous step were combined and loaded on to the colu~n, i.e. a total volume of about 5 ~ of buffer containing about 5 g Apo~ analog. The column wa~ then w~shed with 2.8 CV o~ equilibration buffer.
The first elution was performed with 3 CV of equilibration buffer containing 20 ~M NaCl and the ~eco~d elution was performed with about S.S CV of equilibration buffer containing 40 m~ NaCl. Fractions were collected, monitored and analyz~d as described above, and 2.G CV were combined and saved. The level of endotoxin wa mea~ured by the LAL
assay and was now lecs than 250 pg/mg ApoE analog.

Two subsequent ~lution~ usin~ buffer containing 70 mM NaCl and 350 ~M NaCl re~pectively elut~d the in~rt ApoE analog.

Concentxa~ion and dia ~ 8 ~ter 0-Sepharose The QS-deriv0d saved pooled fractions were concentrated and dialyzed by ultrafiltration through a ~illipore Pellicon Ultrafiltration sy~tem using one lOOK cassette.
The dialysis buffer was lo mM tris/HCl, 1 mM EDTA, 0.1%
TritonQ, pH-7.5. The sample was dialyzed using the recirculating mode whilst maint~ining the ApoE concentration ~t 2-3 ~g/ml. The final retent~te volume was about 500 ml.
F. CM-SEPHAROSE C~ROMATOGRAPHY

2 ~ (j PCr/USg1 /045~3 The purpose of this step i5 to further remov~ endotoxins and to lower the concentration of TritonR to 0.05%.

In this step a 120 ml CM-Sepharo~e Fa~t Flow ~Pharmacia) column was u6ed. The eguilibration buffer was 20 mM Na acetate, 1 mM EDTA, 0.2% TritonR, pH~4.8. After equilibration, the retentate solution ~rom the previous step was loaded on to the CM-Sepharose column. The capacity of the column was 10 mg ApoE/ml and the flow rate was 10 CV/hr.

The column was then washed with the following solutions: 4 CV of equilibration buffer followed by 5 CV of equilibration buffer oo~taining 70 mM NaCl followed b~ 2 CV of 20 mM Na acetate, 1 mM EDTA, 0.05% Triton~, 70 m~ NaCl p~=4. Q . The eluate from the loading and washing ~teps was discarded.

The column was then eluted. The eluent was 8 CV of 20 mM Na acetate, 1 mM EDTA, 0.05% TritonR, 300 mM NaCl, pH=5Ø The progress of the elution was monitored by continu~usly following the abgorbance of the eluate at 280 nm. ~Two different base line~ are used dur~ng the elution: one is the high U.V. absorbance buffer containing 0.2% Triton, the other is the low U.V. ~bsorbance buffer containing-O.05%
Triton. The u~e of a ~n~itivity scale of ~bout 1.0 OD
allows both buffers to appear on the chart column, the low at th~ foot and ~he high at about O.5 OD ~

The ~ample containing the ApoE analog was immediately titrated to pH 7.8 and ~aved. The endotoxin l~vel in this sampl~ wa~ bel~w 50 pg per mg ApoE analog as ~easured by the LAL a-say.

G lOOK ULTRAFILTRATION - TRIT~ REMOY~L
.

The purpose of this step is to remove the TritonR.

W093/~J3 ~q~,6 -38- PCT/US91/04~

This step was carried out at 4C using the Millipore Pellicon Ultrafiltration System, containing one lOOK
cassette, pre-washed w~th 0.5 M NaOH overnight. The flow rate was 9-12 L/hr and the inlet/pre~sure was 5-10 psig.
(This low flow rate is used to prevent aggregation of the ApoE analog as the Triton~ is being removed.) The ApoE
~ample from the previous step (960 ml containing about 600 mg ApoE analog) was diluted to O.S mg/ml with 10 mM NaHCO3 buffer pH=7.7.

The sample was then treated in the ultrafiltration system and the following conditions were applied throughout ~his TritonR removal step:

a) The Triton~ concentration must be lower than 0.02%
i.e. the TritonR concentration must be below its critical micelle concentration in order to achieve effective Triton~ removal acro~s the lOOK membrane.

b) The ApoE analog must not be diluted below 0.5 mg/ml or difisociation of the ApoR molecule will occur and it may cross the 100 R membrane.
, ~
c) The ApoE ~nalo~ ~ust not be concentrated above 1.5 2S mg/ml or aggregation of the ApoE molecules may ~ccur .

The dialysis buffer used in the ultrafiltration system was 10 mM NaHCO3, 150 m~ NaCl, pHz7.8.
After concentration and dilution steps in accordance with the above conditions, the dialy~i~ was performed at constant volume and constant ~low rate and the dialysis was completed when the ab~orbance at 280 nm of the filtrate was 0.01 units. ~Triton~ solution absorbs at 280 nm and an absorbance of 0.01 is equivalent to 0.0005% Triton~.) The ~W093/~3 2 1 ~ 2 2 ~ 6 PCT/US91/~553 '.
total volume of final retentate was 770 ml and the total volume of the filtrate was 9.5 L.

The solution containing the ApoE analog was then filtered (0.2 micron filter) and stored at -700C in 80 ml glass bottles.

OverallLYield:
o.3 g of highly p~rified ApoE analog were recovered from 3 Kg of bacterial cake. The ApoE analog, approximate~y 97%
pure, was in the ~ame aggregation ~tate as plasma ApoE when tested under the ~ame condition~ of gel permeation analysis.
The ApoE analog ~a~ple contained le88 then 50 pg of endotoxins/mg protein.

WOg~ 3 PCT/US91/~
~& -40-Lyo~hilization If the ApoE analog is to be lyophilized the dialysis buffer in the TritonR removal step i5 2 mM NaHC03, pH=7.8, and after lyophilization the samples of ApoE analog are stored at -200C.

After lyophilization the ApoE analog can be redissolved and it retains its normal biological activity. The lyophilized ApoE analog is very stable for at least a year.

2~22~6 ~W093/~3 PCT/US91/04553 ESAMP~ ~

Alternative Im~roved Method For Purification of Re~ombinant ApoE.

This method i8 a proces~ for production of highly purified ApoE analog from the cell ~upernatant and it is suitable for scale-up for industrial application. The general scheme of the downstream process (Scheme II) consists of A' through G' as follows:

A' CELL DISRUPTION - NO MAGNESIUM IONS PRESENT.
B' EXTRACTION OF SUPERNATANT WITH TRITONR.
C' 100K ULTRAFILTRATION.
D' ~EAE CHROMATOGRAPHY
E' HEPARIN-SEPHAROSE CHROMATOGRAPHY
F' CM SEPHAROSE CHROMATOGRAPHY
G' 100X ULTRAFILTRATION - TRITO~ REMOVAL.
Th~ following d~tail~d Qxample of the step~ in purification of the ApoE analog from the supernatant is an example involving 1.5 Xg cell cake. The ~ethod d~scribe~ is suitable for scale-up with only minor ~odifications.
A bac~erial cake weighing 1.5 Kg from on~ or more fermPntation~ was processed downstream ~hrough steps A' to G'. Steps A', B', C' and G' were performed at 4C-10C~
except where otherwise indicated. All other activities were performed at roo~ temperature.

A'. Cell ~isru~tion 1.5 Kg of wet cell cake (containing about 6 g ApoE analog) was suspended in 6 L of buffer EB. (Buffer EB = 50 mM
tris/HCl, 10 mM EDTA, 0.25% beta hydroxybutyrate sodium W093/~3 ~cl~ -42- PCT/US91/~-~3 salt, pH = 7.5~

The suspension was homogenized using a Kinematica homogenizer yielding 7.5 L of homogenate. Disruption was then performed by glass bead grinding (KDL Dynomill) at SL/hr (in two cycles). Three-fold dilution of the resulting suspen~ion using buffer EB yield~d a volume of 22.5 L.

Centrifugation of this lysate was then performed in a continuous CEPA-41 tubular bowl centrifuge, with a feed rate of 9 L/hr. The pellet, weighing approximately 600 g was discarded, and the supernatant (about 22 L) containing the soluble ApoE analog was saved.

B'. Extraction of Cell Pel~et With TritonR

TritonR was added to the supernatant of the previous step to a final concentration of 0.3%. The resultiny suspension was then acidified with HCl to pH s 4.0 and centrifuged on a Sorval centrifuge for S' at 4500 rpm. The pellet weighing about 1,200 g was discarded. Ihe supernatant solut~on (19.8 L), containing the ApoE analog, was titrated to pH 7.5 and saved.

Note: TritonR i5 present in all following steps until it is removed in step G'.

C'. lOOK yltFafil~ration The purpo~e of this step is to remove low molecular weight contaminants by ultracentrifugation/dialysis.

The Millipore Pellicon ultrafiltration system using one lOOK
cassette was utilized to concentrate the supernatant of the previous step to about 12 L. The feed pressure was 20 psig and the dialysis huffex was 20 mM tris/HCl, 1 mM E~A and 2 1 1 2 2 ~ PCT/US91/04553 0.1% TritonR, pH ~ 7 . 5. The 12 L retentate was dialyzed as described in step C, Scheme I; ApoE analog concentration in the retentate was maintained at 2-3 mg/ml. The dialysis yielded about 7.S L of retentate which contained about 5-lO
~g endotoxins per mg ApoE analog a~ measured by the LAL
assay.

D'. ~Ea~Chromatography The purpose of this ctep is to separate the ~poE analog from contaminants ~uch as protein~ and other cellular materials.
In addition, separation of active ApoE analog from inactive ApoE analog and from endotoxins i achieved.

Six runs through a 400 ml column of DEAE Fast Flow column (Pharmacia) were performed. The flow rate waC lO CV/hr.
The capacity of the DEAE colu~n under these conditions was 4 mg ApoElml. The column was fir~t egu~librated with equilibration buffer: 20 mM trislHCl, 1 mM ~DTA, 0.5 Triton~, pH ~ 7.5.

one sixth of the retentate ~olution from step C' was loaded each time and the column was washed with 3 ~ of equilibration ~uff~r. The first elu~io~ W~8 perfor~ed using 3.6 CV of equilibration ~uffer conta~ning 20 mM NaCl and the ~lua~e w~s di~carded. The ~cond elution was performed using about 3.2 CV of ~quilibration buffer containing 80 mM
NaCl. The progE~ss of the run was monitored by continuously following the absorbance of the eluate at 280 D. Fxactions were collected from each run and were analyzed by SDS
polyacryla~ide gel electrophoresis stained by Coomassie Blue. The front of the peak containing a high amount of impurities was discarded and the rest of the peak was sa~ed;
it contained about 100-300 ng endotoxins per mg ApoE analog.

W093/~3 PCT/US91/0~ 3 ~44~

Two ~ubseguent elutions using equilibration buffer containing llO mM NaCl and 2l5 mM NaCl respectively eluted the inert ApoE analog.

Concentration and Analysis After DEAE-Se~harose Th~ saved eluates from the 6 column run~ were pooled (total volume ~ 6.8 L) and dialyzed using the Pellicon ultrafiltration system, with one lO0 K cassette (type PTHK, Nillipore). The dialysis buffer was 20 mM Tris, 1 mM EDTA, 0.1% Triton~, pH = 7Ø The flow rate of the filtrate was 20 L/hr and the inlet pressure was 20 psig. The dialysis yielded about 2 L of retentat~ solution containing the ApoE
ana~og.
E'. Heparin-Sepharose Chromatogra~hY

Heparin-~eph~rose binds ApoE analog effectively. The Heparin- Sepharose was prepared a~ ~escribed by K.H.
Weisgraber and R.W. Mahley. J. Lipid Res. ~L, 316-325 (1980). Th~ purpo~e of this chromatographic step is to pur~fy ApoE analog from E. çoli protein impurities and from - endotoxins, resulting in a ~ery low endotoxin level (~bout 6~ pgtmg). In this ~tep ~nd through to ctep G', half the sample obtained fro~ the previou~ step was processed.

A 300 ml Heparin-Sepharose CL 6B column was used; the capacity at p~ 7~0 is 4 mg ApoE analog/ml Sepharose and the flow rate wa~ about 5 CV/hr.
The equilihration buffer was 20 mM tris/HCl, 1 mM EDTA, 0.2%
TritonS, pH = 7Ø After Qquilibration, the retentate solution from ~tep D' was loaded on to the column, i.e. a total volume of about 1 L of buffer containing about 1 g ApoE analog. The column was then washed with l.5 CV of equilibration buffer and then with 4 CV of pH = 8.o buffer 2~ 12~2~i - ~093/~k~3 PCT/US91/04553 which i~ 20 mM tris/HCl, 1 mM EDTA, 0.2% TritonR, pH 8.o.
The first elution was perform~d with 1.5 CV of pH 8 buffer containing 50 ~N NaCl. The sQcond ~lution was performed with about 1 CV of pH ~ 8.0 buffer containing 500 mM NaCl.

The fractions were monitored and analyz~d a~ described above and the second eluat~ was ~aved, diluted twofold in pH - 8.0 buffer and stored at -20-C. The concentration of endotoxins in thi~ sample was about 60 pg endotoxins per mg ApoE
analog.

Concentratio~ alysis after He~in-Se~harose The stored ~ample was thawed and then concentrated and dialyzed by ultrafiltration through a Millipore Pellicon system, using one 100 X cassette.

The dialysi~ buffer wa~ 10 mM tris/~Cl, 1 mM EDTA, 0.1%
TritonR, pH ~ 7.5. Th~ ~ample wa~ concentrated and dialyzed by repeated dilution and subsequent concentration to maintain th~ ApoE concentration at 2-3 mg/ml. The final retentate volume was saved.
, ~

WO93/~k~3 PCT/US9l/~S3 ~A ~ 6 -46-F'. CM SEPHAROSE C~ROMATOGRAp~Y

The purpose of this step and the conditions of operating it are similar to those of Scheme I, step F, i.e. to remove residual endotoxins, and to lower the concentration of TritonR to 0.05S.

In this step a 120 ml CM Sepharose Fast Flow column (Pharmacia) was used. The eguilibration buffer was 20 mM Na acetate, 1 mM EDTA, 0.2~ TritonR, pH = 4.8. After eguilibration, the retentate solution from the previous step was acidified to pH ~ 4.8 and loaded on to the CM-Sepharose column. The capacity of the column was 10 mg ApoE analog/ml of Sepharose and the flow rate was 10 CV/hr.
The column was then wash~d with the following solutions: 3 CV of equilibration buffer followed by 6.6 CV of eguilibration buffer conti~inin~ 70 m~ NaCl followed by 5 CV
of 20 mM Na acetate, 1 mM EDTA, o.os% TritonR, 70 mM NaCl pH
= 4.8. -~

The column was then eluted. It i5 important for the next step that the ApoE fr~ctions c~lle~ted zre not di~uted below 1 mg/ml. Th~ eluant was about 6 CV of 20 mM Na acetate, 1 ~N EDTA, 0.05~ Triton~, 300 mM NaCl pH = 5Ø
The eluate, containing the Apo~ an~log, was immediately titrat~d to pH = 7.5 and saved; it was ~tored at -2~C. The amount of endotoxins in this s~mple wa~ found to be less than 30 Ps per mg ApoE ~nalog.
G'. Ultrafiltration - Triton~ ~e~oval The purpose of this step, to remove the TritonR, is the same as in Step G, Scheme I (Example 33 and is carried out under similar conditions.

2 1 ~ 2 2 ~ 6 PCT/US91/~553 This step was carried out using the Nillipore Pellicon Ultrafiltration System with one l00 R cassette pre-washed with 0.5 M NaOH overnight. The flow rate was l0 L/hr and the inlet pressure was 5 psig; this lower ~low rate is used to prevent aggregation of the ApoE analog as the Triton~ is being removed. The dilution buffer i8 10 mM NaHCO~ pH = 7.8 The sample was then treated in the ultrafiltration system and the same three condition~ applied throughout this TritonR removal step as recited in step G in Scheme I. The dialysis buffer used in the ultrafiltration system was 2 mM
NaHCO3, pH - 7.8.

After concentration and dilution steps in accordance with the above conditions, the dialysis was performed and was completed when the absorbance at 280 nm of the filtrate was 0.016. The retentate contained about 0.5 mg/ml of ApoE
analog. The solution containing the ApoE analog was then filtered (0.2 micron filter).
Overall Yield: 0.9 g of highly purified ApoE ~nalog were reco~ered from l.5 Xg of bacterial cake. The ApoE analog, approxi~ately 93% pure, was in the same aggregation sta~e as plasmatic ApoE und~r the same conditions of gel permeation analysis and contained le~s than 30 p~ endotoxins per mg.
(~ndotoxin wa~ assayed as described in Exampl~ 3).

The samples of ApoE analog were lyophilized and stored at -20C. This ApoE analog prepared fro~ the c~ll supernatant behaves in a si~ilar fashion after lyophilization to ApoE

W093/~3 ~9 ~ PCT/USgl/0~ -3 analog prepared from the cell pellet. It can be redissolved in water and retains its normal biological activity.

WO93/~L~3 ~ 6 PcT/US9l/n4553 E~A~PLE S

Characterization o~ A~oE Produced By the ImDroved Method Two batches (01 and 02) of ApoE analog produced by two different fermentations and purified by the methods described above in Scheue I (Example 3) were analyzed and compared to naturally occurring ApoE prepared from human plasma (p-ApoE).
A. ,Purity and Homoaeneitv The purity of the r-ApoE was approxi~ately 97% of the total protein as estimated from Cooua~ie blue and silver staining of SDS-PAGE.
:
The protein identified aæ ApoE and which comprises 97% of the total protein includes, in addition to the main band, a band of approxi~ately 10-15% of the total protein that appears just below the ~ain band of ApoE. This band is id~ntified a~ a partially cleaved ApoE as determined by Western blot and by N-terminal amino acid sequence analysis (~ee Section B.5 below).

Residual DNA and ~NA were estimated by measuring the ratio of ab~orbanc~ at 280 to 260 nm in a ~mple of ApoE, using p-ApoE a~ a re~erence ~aterisl (Table ~).

Table I

Sample A2~ /A
p-ApoE 1.74 r-ApoE (8atch 01) 1.74 r-ApoE (Batch 02) 1.78 ~ 50 PCT/US91/~ -S3 The value of the A~/A~ ratios indicates that contamination by nucleic acids i8 below detection limits.

Toxicity The level of bacterial-derived endotoxin was well within the established limits. It was reduced by the purification process to 2S pg/mg ApoE, according to repeated LAL assays.

B. Compa~iso~ o~ r-ApoE to P-AE~~ ``

1. S~-PAGE

Samples from Batch Ol, Batch G2 and p-ApoE were applied on to a 12.5% polyacrylamide-SDS gel in the presence and absence of 2- mercaptoethanol (2ME) as shown in Fig. 4; see Description of the Figure. The main bands for r-ApoE and p-ApoE migrate identically. The low MW band (32 KD) of r-ApoE reprosents a clsavage product of the intact protein (34 KD). The upper MW band that appears in the samples applied without 2ME ~ay represent ~ulfhydryl bonded oligomer of ApoE.

2.
~he ~ntigenic identity of r-ApoE can be determined by Western blot. The conditions and sample preparation of the WeRtern blot are ~imilar to those ~or the SDS-PAGE ~Fig. 4).
After electrophoresis, the gel is blotted onto nitrocellulose and develop~d w~th rabbit polyclonal antiserum specific for human plasmatic ApoE~ An additional band, ~ust abov~ the main band, i~ observed in the plasmatic preparation, but does not appear in the r-ApoE preparations.
This higher molecular weight band probably represents a glycosylated form of p-ApoE.

wo s3/00443 2 ~ 12 2 ~ ~ Pcr/ussl/n4ss3 In the r-ApoE preparations, the low~r (32 KD) band reacts w~th anti~erum and thus r~presents a cleavage product of the molecule (see ~ection 5 below). This low MW band also reacts with anti core Apo-E monoclonal antibodies, but does not react with anti N- Terminus monoclonal antibodies. ~;

The high MW band~ which appear in ~ll Apo-E preparations on the Western blot that are not treated with 2ME, may :
represent multimer~.
3. Moleculdr Weig~ y Size Exçlusion ChromatoaraDhv The ApoE MW and aggregation statQ were determined by fast size exclusion chromatography on Superose 6 column (HR
10/30, Pharmacia). A compari~on of Superose 6 elution profiles ~f r- ApoE and p-ApoE i~ shown in Fig. 1. The MW
of both r-ApoE and p- ApoE were calculated as described in the Description of Fig. 1 to bæ approximately 65 KD thereby indicating a dimer form.
4. Ultra Violet Aksorbance~ S~

- The identity and purity of ~be r-ApoE and the p-ApoE~were also compared by ~ea~uring the W absorbance in the range of 350 to 200 nm, o~ ApoE batch 01 (Fig. 2A) and batch 02 (Fiq.
2B3; these pectra are idantical to the spectrum of p-ApoE
~Fig. 2~ and 2B).

W093/O~U3 ~ 52- pcT/ussl/r S3 5. Amino Acid AnalYsis Amino acid composition analysis of the two batches (ol and 02) of r-ApoE analog and p-ApoE were nearly identical; the s major difference was that the two sample~ of r-ApoE analog contained an add~tional residue of ~ethionine compared to p-ApoE; see Vogel et al., PNAS tUSA) ~: 8676-8700 (1985).

N-terminal ~equence analy~is, 19 cycles, of both batches of r- ApoE analog r~vealed that the N-sequence corresponds to the first 18 amino acids of authentic p-ApoE with an additional N-terminal methionine: there was no evidence in r-ApoE of a secondary sequence without the additional N-terminal methionine.
The 32 KD band which appears just below the main ApoE band (at 34 KD) has b@en isolated and its N-ter~inal sequence analyzed. Th~ result of 14 cycles demonstrated that the N-terminus of this co~poun~ corresponds to residues 12~to 25 of p-ApoE and thQrefore the 32 KD band corre~ponds to ApoE
lacking the first 11 amino acid residues at the N-terminus.

In addition, sa~ple~ of r-ApoE analog before and ~fter lyophilization were c~mpar~d in the following tests and shown to behave virtually identical to one another and similar to auth~ntic p ApoE: SDS-polyac~ylamid~ gel electrophore~is, We~tern blot analysi~, gel filtration and . W spectral analy&is~

~ t ~ ~ 2 2 f?
WO93/~k~3 PCT/US91/~553 C. Bioloaical Activity :

1. Receptor Bindi~q A~sav Phospholipid complexe6 of r-ApoE anslog and dimyri~toyl-phosphatidylcholine (DMPC) w~re pr~par~d and isolated as described by T.L. Innerarity et al., ~t BiQl. Chem. 254:
4186- 4190 (1979).

Lipoprotein receptor binding aæ~ays were per~ormed as described by T.L. Innerarity and R~W. Mahley, iochemist~y 17: 1440-1447 (1978). Iodinations of ApoE were performed in :~
O.10 M NH4HC03 with Iodo-Be~d~ (Pierce) according to the manufacturer'~ directions.
Comparison of the r~ceptor bi~ding of ApoE-DMPC complexes demonstrated that the r-ApoE analog posse~sed binding propertie æimilar to those of the authentic ApoE (p-ApoE).
In these experiments, 6amples of ApoE prepared by Schemes I
and II (~xa~ples 3 ~nd 4 re~pectiYely) were compared to p-ApoE. Table II shows the result~ of one set of competition studies u ing ~ LDL bound to ApoB,E receptors (al~o known a~ low den~ity lipoprotein-LDL-recepto~) on cultured fibrobl~sts. T~s~s u~ing ApoE analog after lyophilization demonstrated that lyophilization had no effect on bioloyical ~ctivity.

W093/~3 PCT/US91/~ -S3 ~ -54-,?.~
Table II
omDetition Studies Usinq 12sI-L~L Bound to A~oB, E Receptors ApoE 50% displacement, ~g/ml p-ApoE 0.050 r-ApoE (Scheme I) 0.033 r-ApoE (Scheme II~ 0.046 2. ~ipoprotein Met~bolis~ in Cultured H~ma~_~ells The approach used was to supplement culture systems (human skin fibroblast and Hep G-2 cells) containing 125I-lipopro~eins with exogenous reco~binant or plasmatic ApoE (ApoE-3). Without adde~ ApoE, cellular metabolism (binding, cell association and degradation) of VLDL
fractions I, II and III was negligibl~, and of IDL about 50%
that of LDL. Exogenous recombinant A~oE analog (before or after lyophilization) cau~ed a ~any-fold e~hanceme~t of VLDL
~etabolism without any appreciable effect on LDL metabolism.

Conclusion ~ 1~

The improved fflethod of APQ~ purification yields a recombinant ApoE analog with properti~s and characteris~ics that closely resemble the propertie of naturally-occurring ApcE isolated from plasma.

211~22~
" ~o 93/00443 PCr/US91/045~3 ~ ' Pharmaceutical and Di~qDQ~tic Uses of ApoE Analog Examples 3 and 4 describe the purification of a novel ApoE
analog whi~h ha~ ~ny potential pharmaceutical, veterinary and diagnostic uses. Some of th~ u~es envisaged for the novel ApoE analog prepared as described in Examples 3 or 4 are described below. The pharmaceutical or veterinary composition containing the ApoE analog should be formulated with a suitable carrier.

Impaired LiDid and Cholesterol Metabolism We envisage administration of the suitably formulated ApoE
analog as purified in Examples 3 and 4 to individuals for therapeutic treatment of atherosclerosis, whether due to dietary or to genetic rea~ons, by lowering blood cholesterol or lipoprotein lev21~. We believe the use of ApoE analog may prevent or have therapeutic effect on ~uch conditions as peripheral vascular disease, atherosclerosis, heart attacks and cerabral va~cular disease.

Another potential u~e of ApoE analog is in the trea~ment of pO8~- myocardial infarction patients. The exogenous admlni~tr~tion of th~ ApoE ~nalog may, in 50~e patients, prevent the re-occlu~ion of the artery whi~h occurs in approximately 30% of myocardial infarction patients who have been treated by angiopl2sty or other t~chniques.

Prophylactic administration of the ApoE analog to prevent atherosclerosis is also considered. This may be especially considered for high risk patients. Example of such patients are those suffering from genetic hyperlipoproteinemia (type ITI hyperlipoproteinemia) due to the occurrence of abnormal w093/~43 ~ ,~3 -56- PCT/~S9l/~ ;3 variant form~ of ApoE that bind poorly to the lipoprotein (LDL) receptors or to the ab~ence (or almost complete absence) of ApoE.

2. ~

We envisage the admini~tration of th~ suitably formulated ApoE analog as purified in Examples 3 or 4 as the therapeutic agent in treatment of damaged neuronal tissue to promote nerve development and regeneration.

3. Treatment of Tumor~ Ex~essina Hiah Lev~ls of LDL
Rece~,oxs We envisage u6ing the suitably formulated ApoE analog as purified in Examples 3 or 4 in a phar~aceutical composition for the treatment of tumors which harbor high levels of LDL
receptors. The ApoE ~ay contain or be linked physically or chemically to a chemotherap~utic or radiotherap~utic agent to produce a target- orientated therap~utic composition.

4. Diaanos~ of_LDL Receptor D~fects , ~
A lipid emulsion containing ~he labeled ApoE analog as purified in Examples 3 or 4 ~ay be u~ed to ~easure the nu~ber of ApoB,E (LDL) receptors, and distribution of uptake of labsl~d ApoE particles. This may possibly be done by scintiscanning technigues, analogous to tho8e used for me~sure~ent of thyroid function. Thece ~e~surements if successful can be used as a diagnosti tool to separate those patiQnt with FH (fam~lial hypercholesterole~ia, i.e.
having absence of, or abnormalities in, the LDL receptors) from hypercholesterolemia patients who do not have this genetic disease.
5. D~aonosis of Primary and Secondary Sites of Tumor 2 ~ 2 ~
PCI`/US91/04~i3 Ç~h :

we envi~age using the ApoE analog, the purif~cat$on of which is describ4d in Example~ 3 and 4, a~ a d~agnostic agent of primary and ~econdary 8ite~ of tumor growth. We particularly envisage the use of a lipid emulsion containing labeled ApoE to local~ze and diagno~e tumor~ harboring high levels of LDL receptors. This may be done by scintiscanning technigues.
6. Immunoreaulation We believe that exogenous administration of the ApoE analog purified as described in Examples 3 or 4 may have therapeutic immunoregulatory activity, and may be used in treatment of autoimmune conditions or diseases involving immunodeficiencies.

7. ipid Emulsions Containing ApoE as Ligand Lipid emul~ion~ hava high af finity for ApoE. We axpect to study the int~raction of ~he Apo~ analog with a variety of lipid Q~ul~ionR and lipo~om~type particl~s. ' This information i~ to b~ ~pplied to th~ ld~ of drug delivery and specific ti88ue- targeting of certain lipid moieties ~uch a~ prostaglandin or leukotriene precursors. Changing thQ lipid composit~on of liposomes has b~en effective in tis~ue targettins; similarly we envisag~ alteration in the ApoE

WO93/OO~ ?~ J6 PCTJUS91/~ i3 . -58-content by addition of the ApoE analog purified as describ~d in Examples 3 or 4 to produce ~i~ilar efects.

~112.~2~
PCT/US91/045~3 ~AKP~ 7 Protection of ApoE From Proteolytic Deara~ation In Vitro ApoE, which has a molecular weight of about 35KD, is susceptible to proteolytic activity both in vivo and 'n vitro (after bactQrial cell di~ruption). The n vitro cleavage in the pre~ence of b~cterial cell extract results in the formation of two polypeptides with approximate molecular weights of 14KD and 21XD.

We surprisingly found that the ~ Yi~o proteolytic degradation of ApoE was reduced dramatically when chemicals such as short chain fatty acidc were added to an assay mix containing purif ied r-ApoE. EDTA also reduced the proteolytic activity.

The in vitro protease activity of the bacterial cell free ex*ract on ApoE wa~ measured using the following 500 ml assay mix which contains:

100 ~1 purified ApoE (25 ~g~.
10-50 ~1 of bacterial cell-frea extract.
50 ~1 chemical 8uch a~ butyric acid or beta-hydroxybutyrate or hexanoic acid (all neutralized) or protea~e inhibitor such as trasylol (aprotinin) or EDTA.
300 ~1 0.1 M tris/acetate buffer pH 6.7~

The bacterial cell-free extract was prepared from cultures of host E. coli cell8 containing no plasmid~ grown at 300C
and heat- shocked for 2 hours at 42C~ 10 0~ quivalents of cells were sonicated in 2 ml of 0.1 M tris/acetate buffer, pH = 6.7.

The reaction mixture was incubated at 42~ for 90 minutes after which 20 ~1 of mixture was removed, brought to 100 ~1 O 93/00443 ~ r~ 6 PCI /US9t /0~ ~3 with 0.1 M tris/HCl buffer pH z 8.0, and 50 ~1 of 3 x SDSgel sample buffer was added. (3 x SDS gel sample buffer contains 187.5 mM tris/HCl pH = 6.8, 2.1 M
beta-mercaptoethanol, 9% SDS, 30~ w/v glycerol and o.s%
s bromophenol blue). 20 ~1 of the resulting mixture was added to slot~ of 12.5% polyacrylamide gels following 10 minutes of heat treatme~t at 100C.

Following Ql~ctrophoresis, the gel~ were tained with Coomassie blue reagent. The intensity of the visualized bands was estimated by a scanner.

The ApoE (34 XD) kand intensities on SDS gels of samples containing no bacterial extract or chemical were used as the controls and compared to thos~ of tre~t~d sampl~. The 14KD
and 21KD bandfi produced a~ a refiult of proteolytic digestion both reacted with anti-Apo~ antibody. However, these 14KD
and 21RD band~ were not generated in the presence of effective amounts of the above~ ted chemical~.
Table IV summarizQs the effQct of a vari~ty of che~icals on the proteolyt~c ac~ivity of bactQrial cell-free extract on ApoE. ~hi~ tabl~ clearly de~on~trate~ ~hat fatty acid~ and al80 EDTA pr@Y~nt the d~gr~ation of ~po~ by bacterial extract. Hexanoic (caproic) acid, ED~A, butyric acid and ~ta-hydroxybu~yrate (all a~ids neutralized) are all pr~ferred inhibitors of thi~ proteolytic activity, in order of incr~a~inq inhibitory activity.

It is envisaged that other fatty acids, fatty acid precursors, triglycerides and triglyceride precursors could also be u~ed to inhibit ApoE degr~dation.

It was decided, for reasons of cost and convenience, to choose beta-hydroxybutyrate (sodium salt) as the protease inhibitor at the time of cell disruption (Examples 3A and ~'V093/~3 ~ 26 PCTIUS ll/o4553 4A). EDTA wa~ also added in the alternat~ve method, Scheme II (Example 4A). The preferr~d concentration of the above inhibitors of protea6e degradation at the time of cell disruption is about 0.1~ - 1.0~ with the most preferred concentration being about 0.2~.

W093/~3 PCT/US91/~ -S3 ~~

Ta~le III

EFFECT OF VARIOUS CHEMICALS ON I~ VITRO ~EGRADATION OF
PURIFIED APOE BY BACTERIAL.EXTRACT

Chemical Bacterial Intact ApoE
(34 KD band~ :
Added Extract Added Remaining None Yes +
None (control) No ++++
EDTA Yes +++
Butyric acid Yes +++
Beta-hydroxybutyricYes ++
Hexanoic acid Yes +++(+) Trasylol Yes +

l. ~+++ = about 100% of control.
+++ = about 75~ of control.
++ - about 50% o~ control.
+ = about 25% of control.

Claims (71)

What is claimed is:

1. A method for obtaining a purified recombinant ApoE
or polypeptide analog thereof from genetically engineered bacterial cells which produce the recombinant ApoE or polypeptide analog thereof which comprises:

(a) culturing the genetically engineered bacterial cells so as to produce the recombinant ApoE or polypeptide analog thereof;

(b) treating the bacterial cells in the presence of magnesium ions so as to obtain a lysate containing insoluble recombinant ApoE or polypeptide analog thereof;

(c) recovering from the lysate insoluble material including insoluble recombinant ApoE or polypeptide analog thereof;

(d) treating the insoluble material so recovered with a solution containing a non-ionic detergent to obtain solubilized recombinant ApoE or polypeptide analog thereof;

(e) treating the solubilized recombinant ApoE or polypeptide analog thereof so as to concentrate and purify the recombinant ApoE or polypeptide analog thereof; and (f) recovering the resultant concentrated purified recombinant ApoE or polypeptide analog thereof.

2. A method of claim 1, wherein the bacterial cells in step (a) are Escherichia coli.

3. A method of claim 1, wherein the treatment in step (b) comprises mechanical disruption.

4. A method of claim 1, where the treatment of step (b) takes place additionally in the presence of an inhibitor of proteolytic digestion of ApoE or polypeptide analog thereof.

5. A method of claim 4 where the inhibitor of proteolytic digestion of ApoE or analog thereof is a neutralized fatty acid.

6. A method of claim 5 where the neutralized fatty acid is beta-hydroxybutyrate.

7. A method of claim 1, wherein the recovery of insoluble material from the lysate in step (c) comprises centrifugation.

8. A method of claim 1, wherein the non-ionic detergent is PEG(9-10)p-t-ocytylphenol.

9. A method of claim 1, wherein the treatment in step (a) to concentrate and purify the ApoE or analog comprises ultrafiltration.

10. A method of claim 9, wherein ultrafiltration removes molecules of molecular weight less than 1 X
105 daltons.

11. A method of claim 1, wherein the treatment in step (e) comprises chromatography.

12. A method of claim 11, wherein the chromatography comprises ion exchange chromatography.

13. A method of claim 12, wherein the ion exchange chromatography is performed using a tertiary amine ligand attached to a resin.

14. A method of claim 13, wherein the resulting concentrated, purified ApoE or analog is dialyzed and the resulting retentate containing further purified ApoE is saved.

15. A method of claim 14, wherein the resulting retentate containing further purified ApoE or analog is highly concentrated and purified by a quaternary amine ligand attached to a resin.

16. A method of claim 15, wherein the resulting highly concentrated, purified ApoE or analog is dialyzed, saving the retentate.

17. A method of claim 16, wherein the resulting highly concentrated, purified ApoE or analog is further concentrated and purified by cation exchange chromatography.

18. A method of claim 17 wherein the cation exchange chromatography is performed using a carboxy methyl-ligand attached to a resin.

19. A method of claim 1, wherein the recovery of the concentrated, purified ApoE or analog in step (f) comprises ultrafiltration.

20. A method of claim 19, wherein the ultrafiltration removes molecules of molecular weight less than 1 X

105 daltons.

21. A method of claim 19, wherein the ultrafiltration removes the non-ionic detergent.

22. A method for obtaining a purified recombinant ApoE
or polypeptide analog thereof from genetically engineered bacterial cells which produce the recombinant ApoE or polypeptide analog thereof which comprises:

(a) culturing the genetically engineered bacterial cells so as to produce the recombinant ApoE or polypeptide analog thereof;

(b) treating the bacterial cells in the presence of EDTA so as to obtain a lysate containing soluble recombinant ApoE or polypeptide analog thereof;

(c) recovering from the lysate a solution containing soluble recombinant ApoE or polypeptide analog thereof;

(d) treating the solution so recovered with a solution containing a non-ionic detergent to obtain a second solution containing solubilized recombinant ApoE or polypeptide analog thereof;

(e) treating the second solution containing recombinant ApoE or polypeptide analog thereof so as to concentrate and purify the recombinant ApoE or polypeptide analog thereof; and (f) recovering the resultant concentrated purified recombinant ApoE or polypeptide analog thereof.

23. A method of claim 22, wherein the bacterial cells in step (a) are Escherichia coli.

24. A method of claim 22, wherein the treatment in step (b) comprises mechanical disruption.

25. A method of claim 22, where the treatment of step (b) takes place additionally in the presence of an inhibitor of proteolytic digestion of ApoE or polypeptide analog thereof.

26. A method of claim 25 where the inhibitor of proteolytic digestion of ApoE or analog thereof is a neutralized fatty acid.

27. A method of claim 26 where the neutralized fatty acid is beta-hydroxybutyrate.

28. A method of claim 22, wherein the recovery of soluble material from the lysate in step (c) comprises centrifugation.

29. A method of claim 22, wherein the non-ionic detergent is PEG(9-10) p-t-ocytylphenol.

30. A method of claim 22, wherein the treatment in step (e) to concentrate and purify ApoE or analog comprises ultrafiltration.

31. A method of claim 22, wherein ultrafiltration removes molecules of molecular weight less than 1 X
105 daltons.

WO 93/00443 PCT/US91/???53

32. A method of claim 22, wherein the treatment in step (e) comprises chromatography.

33. A method of claim 32, wherein the chromatography comprises ion exchange chromatography.

34. A method of claim 33, wherein the ion exchange chromatography is performed using a tertiary amine ligand attached to a resin.

35. A method of claim 34, wherein the tertiary amine ligand is diethyl amino ethyl.

36. A method of claim 35, wherein the resulting concentrated, purified ApoE or analog is dialyzed and the resulting retentate containing further purified ApoE is saved.

37. A method of claim 36, wherein the resulting concentrated, purified ApoE or analog is dialyzed and the resulting retentate containing further purified ApoE or analog is saved.

38. A method of claim 37, wherein the resulting retentate containing further purified ApoE or analog is highly concentrated and purified by affinity chromatography.

39. A method of claim 38 wherein the affinity chromatography is performed using a heparin ligand attached to a resin.

40. A method of claim 39, wherein the resulting highly concentrated and purified ApoE or analog is dialyzed and the resulting retentate containing highly concentrated and purified ApoE or analog is saved.

41. A method of claim 40, wherein the resulting retentate containing highly concentrated and purified ApoE or analog is further concentrated and purified by cation exchange chromatography.

42. A method of claim 41, wherein the cation exchange chromatography is performed using a carboxymethyl ligand attached to a resin.

43. A method of claim 22, wherein the recovery of the concentrated, purified ApoE or analog in step (f) comprises ultrafiltration.

44. A method of claim 43, wherein the ultrafiltration removes molecules of molecular weight less than 1 X
105 daltons.

45. A method of claim 43, wherein the ultrafiltration removes the non-ionic detergent.

46. A composition comprising ApoE or analog thereof produced by the method of claim 1 or 22 and a suitable carrier.

47. A composition comprising ApoE or analog thereof produced by the method of claims 1 or 22 wherein the ApoE or analog thereof contains or is linked physically to a chemotherapeutic or radiotherapeutic or radiodiagnostic agent, and a suitable carrier.

48. A solution of biologically active ultrapure ApoE or analog thereof containing less than 10.0 pg endotoxin per mg which may be lyophilized and retain biological activity on redissolution.

49. A method of treating a subject suffering from atherosclerosis which comprises administering to the subject an amount of ApoE or analog thereof of claim 46 effective to combat atherosclerosis.

50. A method of treating a subject suffering from hypercholesterolemia caused by impaired cholesterol metabolism which comprises administering to the subject an amount of ApoE or analog thereof of claim 46 effective to normalize cholesterol metabolism so as to alleviate hypercholesterolemia and thereby treat the subject.

51. A method of treating a subject suffering from hyperlipoproteinemia caused by impaired lipid metabolism which comprises administering to the subject an amount of ApoE or analog thereof of claim 46 effective to normalize lipid metabolism so as to alleviate hyperlipoproteinemia and thereby treat the subject.

52. A method of treating a subject suffering from neuronal injury which comprises administering to the subject an amount of ApoE or analog thereof of claim 46 effective to promote nerve development and regeneration.

53. A method of treating a subject suffering from a tumor which harbors high levels of LDL receptor which comprises administering to the subject an amount of ApoE or analog thereof of claim 47 effective to treat the tumor.

54. A method of diagnosis of LDL receptor defects in a subject by administering to the subject an amount of ApoE or analog thereof of claim 47 effective to quantitate the LDL receptors.

55. A method of diagnosis of primary or secondary sites of tumor growth in a subject where the tumor harbors high levels of LDL receptors which comprises administering to the subject an amount of ApoE or analog thereof of claim 47 effective to visualize the tumor.

56. A method of treatment of autoimmune disease in a subject which comprises administering to the subject an amount of ApoE or analog thereof of claim 46 effective to treat the subject.

57. A method of treatment of a subject having an immunodeficient disease which comprises administering to the subject an amount of ApoE or analog thereof of claim 46 effective to treat the subject.

58. A lipid emulsion comprising ApoE or analog thereof produced by the method of claims 1 or 22 wherein the ApoE or analog thereof is a ligand.

59. The use of the lipid emulsion of claim 58 for drug delivery and tissue targetting.

60. A method for increasing the production of ApoE or analogs thereof in a bacterial host by adding to a medium in which the host is growing an effective amount of a compound selected from the group consisting of neutralized fatty acids, triglycerides, triglyceride precursors and acetate.

61. The method of claim 60, wherein the neutralized fatty acid is sodium propionate, n-butyric acid or beta-hydroxybutyric acid.

62. The method of claim 60, wherein the triglyceride is triacetin, tributyrin, tricaprylin or glycerol.

63. The method of claim 60, wherein the triglyceride precursor is 1-monomyristoyl-rac-glycerol, 1-monopalmitoyl-rac-glycerol or DL -.alpha.-hydroxy isovaleric acid.

64. The method of claim 60, wherein the acetate is sodium acetate.

65. The method of claim 64 wherein the effective amount of sodium acetate produces a medium concentration of 0.1% to 1% sodium acetate,

66. The method of claim 65 wherein the medium concentration of sodium acetate is about 0.5%.

67. A method of claim 60, wherein the effective amount of fatty acid, triglyceride or triglyceride precursor produces a final concentration in the culture of about 0.1% to 0.5%.

68. A method of claim 67 wherein the final concentration in the culture is about 0.2%.

69. A method of protecting ApoE protein in solution from degradation by adding to the solution an effective amount of a compound selected from the group consisting of neturalized fatty acids, triglycerides, triglyceride precursors, acetate and EDTA.

70. A method of claim 69 where the effective amount of compound added to the solution produces a concentration of compound in the solution of about 0.1% to 0.5%.

71. A method of claim 70 where the concentration of compound in the solution is about 0.2%.