Purification of molecules

Details Purification of molecules Purification of molecules

1998-10-08

2001-07-24

Low, Christopher S. F. (Department: 1654)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Separation or purification

C530S412000, C530S415000, C530S416000, C210S656000

Reexamination Certificate

active

06265542

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to an improved method for purifying molecules such as peptides, polypeptides, and organic molecules from variants, impurities, and contaminants associated therewith.
2. Description of Related Art
The production of large quantities of relatively pure, biologically active molecules is important economically for the manufacture of human and animal pharmaceutical formulations, enzymes, and other specialty chemicals. For production of many polypeptides and proteins, recombinant DNA techniques have become the method of choice because large quantities of exogenous proteins can be expressed in bacteria and other host cells. The expression of proteins by recombinant DNA techniques for the production of cells or cell parts that function as biocatalysts is also an important application.
Producing recombinant protein involves transfecting host cells with DNA encoding the protein and growing the cells under conditions favoring expression of the recombinant protein. The prokaryote
E. coli
is favored as host because it can be made to produce recombinant proteins in high yields. Numerous U.S. patents on general bacterial expression of DNA encoding proteins exist, including U.S. Pat. No. 4,565,785 on a recombinant DNA molecule comprising a bacterial gene for an extracellular or periplasmic carrier protein and non-bacterial gene; U.S. Pat. No. 4,673,641 on co-production of a foreign polypeptide with an aggregate-forming polypeptide; U.S. Pat. No. 4,738,921 on an expression vector with a trp promoter/operator and trp LE fusion with a polypeptide; U.S. Pat. No. 4,795,706 on expression control sequences to include with a foreign protein; and U.S. Pat. No. 4,710,473 on specific circular DNA plasmids.
Genetically engineered biopharmaceuticals are typically purified from a supernatant containing a variety of diverse host cell contaminants. Reversed-phase high-performance liquid chromatography (RP-HPLC) is commonly used for protein purification because it can efficiently separate closely related protein impurities. Procedures utilizing RP-HPLC have been published for many molecules. McDonald and Bidlingmeyer, “Strategies for Successful Preparative Liquid Chromatography”,
Preparative Liquid Chromatography
, Brian A. Bidlingmeyer (New York: Elsevier Science Publishing, 1987), vol. 38, pp. 1-104; Lee et al., Preparative HPLC. 8th Biotechnology Symposium, Pt. 1, 593-610 (1988). Irreversible binding of insulin and proinsulin to C18 stationary phases has recently been reported (Linde and Welinder,
J. Chromatoqr
., 536: 43 (1991)), with the C4 alkyl chain substitution being preferred to maximize product recovery. Nice et al.,
J. Chromatoqr
., 218: 569 (1981).
Acetonitrile, ethanol, methanol, and isopropanol are often used as eluents for reversed-phase chromatography, and acetonitrile is the most common eluent for this purpose because it produces high-resolution separations. Acetonitrile is used at large scale for purification of recombinant proteins such as insulin. Kroeff et al.,
J. Chromatography
, 461: 45-61 (1989). However, acetonitrile and the other common solvents are flammable with all the attendant difficulties, and acetonitrile has a denaturing effect.
Recombinant human insulin-like growth factor-I (rhIGF-I) is a 70 amino acid protein with a pI of 8.4 (Rinderknecht and Humbel,
Proc. Natl. Acad. Sci. USA
, 73: 2365 (1976); Rinderknecht and Humbel, 253: 2769-2776 (1978)) and with a molecular weight of 7649 daltons and three disulfide bonds. Raschdorf et al.,
Biomedical and Environmental Mass Spectrometry
, 16: 3-8 (1988).
IGF-I has been purified by RP-HPLC from human plasma (Cornell et al.,
Preparative Biochemistry
, 14: 123-138 (1984); Petrides et al.,
Endocrinology,
118: 2034-2038 (1986)) and from recombinant material produced in bacterial fermentation. Olson et al.,
J. Chromatography
, A675: 101-112 (1994). See also U.S. Pat. No. 5,446,024 on purifying IGF-I using RP-HPLC, as well as Svoboda et al.,
Biochemistry
, 19: 790 (1980); Cornell and Brady,
J. Chromatogr
., 421: 61 (1987); and Francis et al.,
Endocrinology
, 124: 1173 (1989).
RP-HPLC can separate several variant forms of IGF-I, including met
59
O variant (methionine sulfoxide at position 59, identified by Hartmanis and Engstrom,
Techniques in Protein Chemistry
, 327-333 (1989)), desGly
1
desGly
1
Pro
2
variant (N-terminal glycine and proline missing), carbamylated variant (chemistry of carbamylation in Qin et al.,
J. Biological Chemistry
, 267: 26128-26133 (1992)), and IGF-I aggregates. During HPLC purification of IGF-I, variants must be removed to historical levels, which includes a requirement of less than 2% met
59
O variant. Purity is determined by a VYDAC™ HPLC assay, which is similar to the assay characterized by Canova-Davis et al.,
Biochem. J
., 285: 207-213 (1992). The amounts of each variant can change from batch to batch.
Olson et al., supra, designed parameters for maximum separation of met
59
O variant from IGF-I, with a buffer of 100 mM potassium phosphate at pH 7.0 and elution with acetonitrile. A typical batch size for the HPLC purification step is 12 kg of IGF-I. If the acetonitrile process were scaled directly to the 60-cm diameter column, it would require five cycles to process the batch, for a total processing time of 13 hours. Average recovery yield for the acetonitrile process, calculated as the mass of IGF-I in the purified pool divided by the mass of IGF-I loaded (mass determined by the VYDAC™ assay), is about 80%, and throughput is about 0.3 g hr
−1
cm
−2
.
There is a need in the art for an efficient reversed-phase liquid chromatography protocol for selectively separating molecules such as peptides, polypeptides, and non-peptidyl compounds from other molecules using a solvent that is less toxic, less expensive, less denaturing, and less flammable than flammable solvents often used as eluents for reversed-phase chromatography, such as acetonitrile, ethanol, methanol, and isopropanol. In particular, there is a need for purifying IGF-I from hydrophobic polypeptides in a fermentation broth, particularly since typically the final process pool contains several variant species of IGF-I that are difficult to separate. This need would be satisfied when the process duplicates as much as possible the yield, purity, throughput, and operating conditions of the liquid chromatography process wherein elution is conducted by a flammable solvent such as acetonitrile.
SUMMARY OF THE INVENTION
This invention provides, in one aspect, a process for purifying a molecule selected from the group consisting of a peptide, a polypeptide, and a biologically active non-peptidyl compound comprising loading a mixture containing the molecule onto a reversed-phase liquid chromatography column and eluting the molecule from the column with a buffer containing hexylene glycol.
While ethanol, methanol, isopropanol, and, in particular, acetonitrile, often provide good protein separations using reversed-phase liquid chromatography, they are flammable solvents (acetonitrile has a flashpoint of about 15° C.), and using them at large scale requires expensive nonflammable-capable equipment and facilities. Further, acetonitrile is somewhat of a denaturant and is toxic to the environment. The method herein was developed to purify molecules by reversed-phase liquid chromatography using the non-flammable eluent hexylene glycol rather than a flammable eluent. Hexylene glycol, with a flashpoint of about 93° C., produced essentially the same yield, purity, and throughput as acetonitrile and with less denaturing effect. Thus, hexylene glycol may be advantageous as an eluent for, e.g., full-length antibodies and some glycosylated proteins, which have a tendency to be denatured when eluted from a reversed-phase liquid chromatography column. Further, hexylene glycol is less toxic to the environment than certain flammable solvents such as acetonitrile and is available in large quantities in USP grade. Also, hexylene glycol was found to be a better eluent for sam

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