Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Hormones – e.g. – prolactin – thymosin – growth factors – etc.
Reexamination Certificate
2001-11-02
2003-05-13
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Hormones, e.g., prolactin, thymosin, growth factors, etc.
C530S412000, C530S418000, C530S422000, C530S425000, C530S426000
Reexamination Certificate
active
06562951
ABSTRACT:
BACKGROUND OF THE INVENTION
Somatotropins are growth hormones which were originally discovered in pituitary gland extracts of various animals. Recombinant DNA technology has permitted the expression of somatotropins as heterologous proteins from various host cells. Such recombinant somatotropins, e.g., somatotropins produced in a microorganism such as
E. coli
bacteria that has been transformed using recombinant DNA, are typically produced by the host cell in a precipitated, denatured state having reduced or substantially no bioactivity. The absence of bioactivity is generally attributed to the conformation of the recombinant somatotropin molecule, which lacks the formation of disulfide bonds. Recombinant somatotropins are believed to be produced by the host cell in substantially reduced form (without disulfide linkages) due to the relatively high redox potential of host cells such as the
E. coli
cell. Most recombinant somatotropins, such as bovine (bST) and porcine somatotropin (pST), are packaged in the host cell as inclusion bodies, also referred to as refractile bodies, which are cytoplasmic aggregates containing the recombinant somatotropin and oligomers thereof.
In order to recover the recombinant somatotropin in a bioactive state, the somatotropin, e.g., in the form of inclusion bodies, is preferably but not necessarily isolated from the host cell, after which the somatotropin may be solubilized to form somatotropin monomers, which may then be naturated into a bioactive conformation. The naturated somatotropin may then be further purified to remove impurities such as other somatotropin species, e.g., oligomers such as dimers, and host cell proteins, for example, by ion exchange to precipitate the impurities (e.g., as described in U.S. Pat. No. 5,182,369, which is incorporated herein by reference) or other suitable techniques.
For example, Bentle et al., U.S. Pat. No. 4,652,630, which is incorporated herein by reference, refers to a method for solubilization and naturation of somatotropin protein from inclusion bodies using an aqueous solution of urea to solubilize the inclusion bodies containing the recombinant somatotropin. Bentle et al. reports using urea solutions having concentrations ranging from 2.5 to 7.5 M and a pH between about 9 and 12 for the solubilization step. Once solubilized, the somatotropin protein can be naturated according to the Bentle et al. method at an alkaline pH.
Other methods have used a detergent to solubilize and naturate a somatotropin. For example, European Patent Specification publication nos. 229,110 and 263,902 (The Upjohn Co.), which are incorporated herein by reference, disclose a method for converting an insoluble form of somatotropin from a transformed microorganism to the native disulphide bond conformation by solubilizing and oxidizing the somatotropin in the presence of a detergent. That method uses a detergent of sodium dodecylsul fate (SD S) or a detergent of the formula:
CH
3
—(CH
2
)
n
—CO—NR
1
—CHR
2
—COOH,
wherein n is 8 through 20 inclusive; R
1
is methyl or ethyl; and R
2
is hydrogen, ethyl, methyl, n-propyl or isopropyl. These specifications further disclose a particular method for solubilizing recombinant bST using an aqueous solution of N-lauroyl methyl glycine, which is represented by the formula:
CH
3
—(CH
2
)
10
—CO—NR
1
—CHR
2
—COOH,
in a sodium borate buffer at 0.1 to 0.5 M and a pH of 8 to 10.5. After solubilization and naturation, European Patent Specification publication nos. 229,110 and 263,902 state that the detergent is removed by an anion exchange resin.
Other publications also disclose methods for the solubilization and naturation of recombinant somatotropins using various detergent and non-detergent compounds including U.S. Pat. Nos. 4,677,196 and 4,766,224, which also use SDS; U.S. Pat. No. 5,023,323, which uses SDS in combination with a chaotropic agent such as urea or guanidine hydrochloride; U.S. Pat. No. 5,240,834, which uses sarkosyl (N-lauroyl sarcosine), and U.S. Pat. No. 4,975,529, which uses 2-amino-2-methyl-1-propanol. Each of these patents are incorporated herein by reference. Although SDS has been reported in several references for use in somatotropin solubilization and naturation methods, it is further acknowledged that SDS binds relatively tightly to the naturated somatotropin, thus making its complete removal from the somatotropin solution difficult.
There is a need in the art for more economical and efficient methods to obtain recombinant somatotropins with high yield and purity. In particular, there is a need for methods for the solubilization and naturation of recombinant somatotropin proteins to obtain the somatotropin molecules in a bioactive state, preferably using a low amount of detergent that is readily biodegradable. There is a further need for such methods that use a detergent that is easily removed from the naturated somatotropin.
SUMMARY OF THE INVENTION
This invention generally relates to methods for producing biologically active recombinant somatotropins. More particularly, this invention relates to methods for the solubilization and naturation of recombinant somatotropins comprising contacting a somatotropin with a detergent composition and water under conditions effective to obtain a naturated somatotropin, wherein the detergent composition comprises a C
10
to C
18
acyl glutamate, a C
10
, C
14
or C
18
alkyl sulfate, an alcohol ethoxy sulfate, lauroyl ethylenediaminetriacetic acid (LEDA), a C
10
to C
18
linear alkyl benzene sulfonate, diphenyl disulfonate or an acyl amino acid of formula (I) or formula (II):
wherein A is CH
2
CH
2
CO
2
H, CH
2
CH
2
SCH
3
, CH
2
CO
2
H, CH
2
CONH
2
, CH
2
CH
2
CONH
2
CH(CH
3
)
2
, CH
2
CH(CH
3
)
2
, CH(CH
3
)CH
2
CH
3
, CH
2
C
6
H
5
, CH
2
C
6
H
4
OH or CH
2
OH and mis is an integer from 8 to 16;
CH
3
(CH
2
)
m
CONH(CH
2
)nCO
2
H (II)
wherein n is an integer from 1 to 5 and m is an integer from 8 to 16.
In a further preferred embodiment, the invention is directed to a method for the solubilization and naturation of a recombinant somatotropin using low amounts of biodegradable detergents that are easily removed from the naturated somatotropin by diafiltration, such as a C
10
or C
12
acyl glutamate, N-lauroyl sarcosine, N-decylsulfate (NDS) or lauroyl ethylenediamine-triacetic acid (LEDA).
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention is directed to a method for the naturation of a somatotropin comprising the step of contacting a somatotropin with a detergent composition and water under conditions effective to obtain a naturated somatotropin, wherein the detergent composition comprises a C
10
to C
18
acyl glutamate, a C
10
, C
14
or C
18
alkyl sulfate, an alcohol ethoxy sulfate, lauroyl ethylenediaminetriacetic acid (LEDA), a C
10
to C
18
linear alkyl benzene sulfonate, diphenyl disulfonate or an acyl amino acid of the formula (I) or (II):
wherein A is CH
2
CH
2
CO
2
H, CH
2
CH
2
SCH
3
, CH
2
CO
2
H, CH
2
CONH
2
, CH
2
CH
2
CONH
2
, CH(CH
3
)
2
, CH
2
CH(CH
3
)
2
, CH(CH
3
)CH
2
CH
3
, CH
2
C
6
H
5
, CH
2
C
6
H
4
OH, or CH
2
OH and ms is an integer from 8 to 16;
CH
3
(CH
2
)
m
CONH(CH
2
)
n
CO
2
H (II)
wherein n is an integer from 1 to 5 and m is an integer from 8 to 16. The detergent composition can also comprise an N-alkylated derivative of formula (I) or (II).
The invention is further directed to a method for the solubilization and naturation of a somatotropin comprising the steps of contacting a somatotropin with a detergent composition and water under conditions effective to solubilize the somatotropin, and subsequently adjusting the pH of the resulting somatotropin solution to naturate the somatotropin, wherein the detergent composition comprises a C
10
to C
18
acyl glutamate, a C
10
, C
14
or C
18
alkyl sulfate, an alcohol ethoxy sulfate, lauroyl ethylenediaminetriacetic acid (LEDA), a C
10
to C
18
linear alkyl benzene sulfonate, diphenyl disulfonate or an acyl amino acid of the formula (I) or (II):
wherein A is CH
2
CH
2
CO
2
H, CH
2
CH
2
SCH
3
, CH
2
CO
2
Ballinger Jessica Marie
Storrs S. Bradley
Tou Jacob S.
Beck, Esq. George R.
Howrey Simon Arnold & White , LLP
Low Christopher S. F.
Mohamed Abdel A.
Monsanto Technology LLC
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