Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~ 93/~3 21~ 2 2 ~ G PCTfUS91/~5~3
-1-
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
-5-
biological activity on sub~equent dissolutiQn, and we have
demonstrated that the lyophilized ApoE is very stable.
W09 ~ , 6 ` PCT/US91/0~-~3
-6-
~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~,
2S
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
-13-
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
-17-
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
--19--
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
2 1 1 2 2 2 6 PCT/US91/04553
-21-
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
-23-
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
-27-
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
--28--
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
-29-
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
-31-
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
A CELL DISRUPTION IN PRE5ENCE OF MAGNESIUM
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
-35-
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
-37-
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
-39-
'.
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
-41-
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
-43-
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
-45-
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
-48-
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
-53-
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
--55--
~ '
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
-57-
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
-59-
~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
6 0 -
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
-62-
~~
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.
