Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-02-11
2001-04-10
Huff, Sheela (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S023530, C530S387300, C435S069600, C435S069700, C435S328000
Reexamination Certificate
active
06214984
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to nucleic acid encoding, and methods for preparing, an antibody comprising a linear V
H
—C
H
1—V
H
—C
H
1 heavy chain fragment associated with two chains.
2. Description of Related Art
Hydrophobic interaction chromatography (HIC) is a useful tool for separating molecules based on their hydrophobicity. Generally, sample molecules in a high salt buffer are loaded on the HIC column. The salt in the buffer interacts with water molecules to reduce tne solvation of the molecules in solution, thereby exposing hydrophobic regions in the sample molecules which are consequently adsorbed by the HIC column. The more hydrophobic the molecule, the less salt needed to promote binding. Usually, a decreasing salt gradient is used to elute samples from the column. As the ionic strength decreases, the exposure of the hydrophilic regions of the molecules increases and molecules elute from the column in order of increasing hydrophobicity. Sample elution may also be achieved by the addition of mild organic modifiers or detergents to the elution buffer. HIC is reviewed in
Protein Purification,
2d Ed., Springer-Verlag, New York, pgs 176-179 (1988).
HIC has been used by various researchers for purification of antibodies. Danielsson et al.,
Journal of Immunological Methods
115:79-88 (1988) found that HIC was particularly useful for purification of monoclonal antibodies from mouse ascites when the isoelectric point of the antibodies was below 7.2. HIC was performed with an Alkyl SUPEROSE HR™ column. The buffer system was 0.1 M phosphate, with addition of ammonium sulfate. Usually the starting buffer contained 2 M ammonium sulfate. Bridonneau et al.
Journal of Chromatography
616:197-204 (1993) were interested in determining whether or not different HIC columns could be used for selective purification of human immunoglobulin G (IgG) subclasses. The antibodies were adsorbed on Phenyl-, Butyl-, or Octyl-SEPHAROSE™ columns in 1 M ammonium sulfate (pH 7.0) and eluted with decreasing salt gradient. Octyl-SEPHAROSE™ medium yielded a poorly adsorbed fraction somewhat enriched in IgG
2a
. See also Berkowitz et al.,
Journal of Chromatography
389:317-321 (1987); Gagnon et al. (90th Annual Meeting, American Society for Microbiology, Anaheim, May 13-17, 1990) Abstract No.0-4; Johansson et al.
Biol. Recombinant Microorg. Anim. Cells,
(Oholo 34 Meeting), 409-414 (1991); Pavlu et al.,
Journal of Chromatography
359:449-460 (1986) and Abe et al.,
Journal of Biochemical and Biophysical Methods
27:215-227 (1993) concerning HIC of antibodies.
HIC has also been used for purifying antibody fragments. Inouye et al.,
Protein Engineering,
pgs 6, 8 and 1018-1019 (1993); Inouye et al.,
Animal Cell Technology: Basic
&
Applied Aspects
5:609-616 (1993); Inouye et al.,
Journal of Biochemical and Biophysical Methods
26:27-39 (1993) and Morimoto et al.,
Journal of Biochemical and Biophysical Methods
24:107-117 (1992) prepared F(ab′)
2
fragments from pepsin digests of mouse IgM monoclonal antibodies using a TSKgel Ether-5PW™ HIC column. The antibody fragments were salted out with 60% ammonium sulfate and the precipitates were dissolved into phosphate-buffered saline (PBS, pH7.4) containing 1 M ammonium sulfate. This solution was loaded onto the HIC column which had been equilibrated with PBS also containing 1 M ammonium sulfate. The F(ab′)
2
fragments which were adsorbed onto the column were eluted by reducing the ammonium sulfate concentration in the elution buffer to 0 M. Inouye et al., found that the fraction containing the F(ab′)
2
was homogeneous by both SDS-PAGE and gel filtration HPLC. The method was considered to be suitable for large-scale purification of F(ab′)
2
fragments. Similarly, Rea et al.,
Journal of Cell. Biochem.
Suppl. 0, Abstract No. X1-206 (17 Part A), p. 50 (1993) evaluated HIC for purification of a F(ab′)
2
fragment produced by peptic digestion of a murine IgG
2a
monoclonal antibody. Protein A purification for removal of residual intact antibody preceded the HIC step. The purification performance of three different HIC columns was tested at several different salts and pHs. POROS PE™ (Phenyl ether) was found to be the best column and phosphate-buffered sodium sulfate at pH 8 gave the best resolution of the F(ab′)
2
fragment.
SUMMARY OF THE INVENTION
In contrast to the above described HIC techniques, which are generally performed at about neutral pH in the presence of high salt concentrations (using a salt gradient to elute the antibody), the instant invention relates to low pH hydrophobic interaction chromatography (LPHIC) for antibody purification. Preferably, the LPHIC is performed at low salt concentrations, i.e., about 0-0.25 M salt, preferably about 0-0.1 M salt and more preferably 0-50 mM salt. This low salt concentration also applies to the loading buffer. Preferably, no salt gradient is used to elute the antibody.
In particular, the invention provides a process for purifying an antibody from a contaminant which comprises loading a mixture containing the antibody and the contaminant on a hydrophobic interaction chromatography column and eluting the antibody from the column with a buffer having a pH of about 2.5-4.5. Preferably the buffer is at a pH of about 2.8-3.5 and more preferably at a pH of about 3.1. Usually, the mixture loaded onto the column is at about the same pH as the elution buffer.
The method is particularly useful for purifying antibody fragments, especially correctly folded and disulfide linked antibody fragments (e.g. Fab fragments) from contaminating antibody fragments which are not correctly folded and/or disulfide linked. The invention resides, at least in part, in the identification of a problem associated with the formation of recombinant immunoglobulins. It has been observed that such production results in the formation of functional F(ab′)
2
antibodies as well as a variety of incorrectly associated light and heavy fragments. The most difficult impurity to remove has been characterized herein as a correctly folded antibody fragment whose light and heavy chains fail to associate through disulfide bonding. This antibody impurity can be detected by SDS PAGE gels and Reverse Phase HPLC as heavy and light chains. The LPHIC described herein provides a means for substantially removing this contaminant from partially purified compositions derived from host cells producing the recombinant antibody fragment, although it is not limited to purification of recombinant products.
The invention also relates to the antibody formulation prepared by the process and uses for this antibody formulation.
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Abe et al., “Purification of monoclonal antibodies with light-chain heterogeneity produced by mouse hybridomas raised with NS-1 myelomas: application of hydrophobic interaction high-performance liquid chromatography”Journal of Biochemical and Biophysical Methods27:215-227 (1993).
Alfthan et al., “Purification of Labelled Antibodies by Hydrophobic Interaction Chromatography”J. Chromatography470 (385-389):385-389 (1989).
Axelsson K., “Bacterial Lipopolysaccharides and Glutathione Mixed Disulfides as Possible Contaminants of Human Growth Hormone Produced with the Use ofE. coliK12”Acta Chemica Scandinavica, Series B39(1):69-77 (1985).
Berkowitz et al., “Use of high-performance hydrophobic interaction chromatography for the determination of salting-out conditions of proteins”Journal of Chromatography389:317-321 (1987).
Bridonneau et al., “Behaviour of human immunoglobulin G subclasses on thiophilic gels: comparison with hy
Genentech Inc.
Helms Larry R.
Huff Sheela
Lee Wendy M.
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