Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1999-12-17
2001-04-10
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S350000, C530S383000, C435S069100, C435S069600, C435S070100, C435S813000, C435S814000
Reexamination Certificate
active
06214975
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field
The present invention relates generally to the production of target proteins in cell culture, and specifically relates to a method of assaying a sample of the cell culture medium to evaluate the quality of the target protein in the sample.
2. Background
Blotting procedures have been cited in the literature since 1975 when Southern published his method of DNA fragment identification after transfer of the DNA from a gel to a nitrocellulose membrane (Southern, 1975). This type of macromolecular transfer was followed by the transfer of RNA onto a filter matrix, which was termed Northern blotting (Alwine et al., 1977). The transfer of proteins from gels onto membranes occurred in 1979 and was termed Western blotting (Towbin et al., 1979). Since then, numerous reviews have been written describing protein blotting (Dunbar, 1994; Gershoni, 1988). The immobilization and detection of proteins generally involves five basic steps. The first step is the electrophoresis of the proteins on a PAGE-gel. Next, the proteins are transferred from the gel and immobilized onto a membrane. Third, non-specific sites on the membrane are blocked so as to increase the signal to noise ratio. The fourth step includes the binding of specific antibodies to the immobilized proteins and subsequent binding of a secondary antibody that recognizes the primary antibody. Finally, the secondary antibody is detected via a detection system, typically involving an enzymatic reaction.
The four main detection systems used on immunoblots are radiometry, calorimetric, bioluminescence, and chemiluminescence. Radiometry involves labelling the samples/antibodies with radioisotopes and exposing the blot to autoradiography film. Although this procedure is sensitive it requires the handling and disposal of radioisotopes and a radioactivity safe facility. Furthermore, the exposure time to the X-ray film is much longer than that for other detection methods. Colorimetric detection methods involve using a secondary antibody that is conjugated to an enzyme, for example alkaline phosphatase, which will react with a colored substrate such as bromochloroindolyl phosphate and nitroblue tetrazolium, to produce a color wherever the antigen/primary antibody complex has reacted with the secondary antibody. The colorimetric method is more sensitive and faster than the radioactive method but does not give a permanent hard copy and is not as sensitive as chemiluminescence or bioluminescence.
More recently, detection systems that have focused on the detection of light have become more common because of their high sensitivity and prolonged and rapid signal output. Bioluminescence and chemiluminescence are the two most sensitive detection methods used for Western blotting). Although both involve the emission of light, they primarily differ in the substrate they use. Bioluminescence substrates such as luciferin are natural products while chemiluminescent substrates such as luminol are made synthetically. Bioluminescence detection involves the release of activated luciferin from luciferin-o-&bgr;-galactoside by &bgr;-galactosidase during its interaction with an alkaline phosphatase conjugated secondary antibody. The luciferin is oxidized to oxyluciferin by luciferase, with light being a product of the reaction. This technique has allowed the detection of as little as 5 fg of protein (Geiger, 1994).
Chemiluminescence involutes the oxidation of a peracid salt by horseradish peroxidase (HRP) that is conjugated to a secondary antibody. This oxidation reaction raises the oxidation state of the HRP heme group. As the electron attempts to come back to its ground state it reacts with lumincil to form a luminol radical. As the luminol radical decays it emits light, which is then detected on an autoradiography film. The use of enhancers can prolong the luminol decay for up to 24 hours, which is the main advantage of chemiluminescence over bioluminescence. Chemiluminescence is as sensitive and rapid (often only a few seconds of film exposure is necessary) as bioluminescence. The sensitivity of the chemiluminescence can be significantly improved if the secondary antibody has been conjugated with biotin. This complex can then be exposed to avidin conjugated HRP conjugate and reacted with the luminol reagents. Chemiluminescence is a preferred method of detection because of its high sensitivity, its rapid speed of detection, its prolonged emission time as well as the ready availability of reagents in a kit format from a large number of suppliers.
Common applications of Western blotting include immunodetection of antigenic sites on polypeptides and for amino acid sequencing. Other applications for Western blots include the detection and characterization of glycoprotein carbohydrate chains (Sato et al., 1998) and detection of receptors zand the study of protein-protein interactions. This has all been made possible by the extensive and sensitive detection methods used in immunoblotting. Most applications, however, have been confined to confirming the presence or absence of product in cell extracts and not for assessing the overall quality of the product for the prediction of downstream purification yields. (Kennel et al., 1998).
SUMMARY OF THE INVENTION
A sensitive, generic, electrophoretic based method has now been developed for assessing the quality and the potential for purification of target proteins directly from fermenter harvests, without any need for concentrating the sample. This has been demonstrated with recombinant Factor VIII (rFVIII) produced by a BHK (baby hamster kidney) cell line.
In a preferred embodiment the technique involves running a desalted sample of growth medium on SDS-PAGE followed by Western blot assay using antibodies which bind the target protein, e.g. anti-Factor VIII monoclonal antibodies. This technique provides a significant improvement in sensitivity over conventional Western blot detection techniques. A similar approach has been used previously to characterize proteolytic fragments of rFVIII during cell culture (Kaufmnan et al., 1988). Routine detection as low as 4 ng of target protein has been accomplished by utilizing chemiluminescent substrates in combination with an amplification system having an anti-mouse biotinylated secondary antibody that reacts with avidin-labelled horse-radish peroxidase. Furthermore, this assay has a high throughput and therefore is particularly useful during fermentation development for rapid screening of multiple fermenter conditions and ultimately for determining the potential for purification. The assay has also been applied to the successful prediction of purification process yield.
We have quite surprisingly found that the chemiluminescent Western blot method (ZAP method) correlates well with other assays which can be more difficult and time consuming to perform. The ZAP method described herein is thus particularly useful as an alternative to those assays (since the only method of monitoring rFVIII in the fermenter is by activity titer). The ZAP method can be correlated with purification yields and can be used to demonstrate that the usual method of monitoring rFVIII (activity titer) is not predictive of product quality. It is not the purpose of this report to assign a cause for any of the examples indicated below but rather to demonstrate that the ZAP method can be a sensitive tool under a variety of conditions. A qualitative classification system was designed and used in conjunction with the ZAP method to categorize the quality of a fermenter and its harvest for monitoring purposes. Furthermore, the ZAP method can be used in conjunction with the qualitative classification system to determine the potential of the harvest for purification processing, without having to actually carry out the purification on each sample.
The purification process can be any optimized series of steps to provide a substantially pure product, i.e., particularly free of cellular contaminants, preferably resulting in a product which is at least 60% pure, preferably at least 80% pure,
Mazer Jonathan
Olson Charles
Park Hyun J.
Zachariou Michael
Bayer Corporation
Beck Michael J.
Giblin James A.
Low Christopher S. F.
Schnizer Holly
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