Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-03-19
2002-12-24
Russel, Jeffrey E. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C502S402000, C530S413000, C530S417000
Reexamination Certificate
active
06498236
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for isolation or purification of immunoglobulins from various raw materials and solid phase matrices therefor.
BACKGROUND OF THE INVENTION
Immunoglobulins—or antibodies—constitute a very important class of proteins which are present in various body fluids of mammals, birds and fish functioning as protective agents of the animal against substances, bacteria and virus challenging the animal. Immunoglobulins are typically present in animal blood, milk, and saliva as well as other body fluids and secretions.
The biological activity, which the immunoglobulins possess, is today exploited in a range of different applications in the human and veterinary diagnostic, health care and therapeutic sector.
Diagnostics
Antibodies have for many years been applied as an important analytic tool in connection with A detection and quantification of a large variety of substances of relevance in the diagnosis of diseases and are increasingly important in areas such as quality control of food products, environmental control, drugs of abuse, and monitoring and control of industrial processes.
For these purposes, the desired antibodies can be produced by hyper-immunisation of suitable host animals, such as rabbits and sheep, or, alternatively, by producing monoclonal antibodies in hybridoma cell cultures.
Following the primary production of the antibodies in either a host animal or in cell culture, the antibody is typically isolated from the bulk of other substances in the raw material by some sort of isolation process. This is necessary in order to avoid interference from these other substances with the antibody activity in the analytical application.
Health Care and Therapeutic Applications
Passive immunisation by intramuscularly injection of immunoglobulin concentrates is a well-known application for temporary protection against infectious diseases, which is typically applied when people are travelling from one part of the world to the other. The success of this kind of treatment on humans is now being followed up in the veterinary field where passive immunisation of new born cattle, horses, pigs and chickens are being applied and developed to enhance the survival rate of these animals during their first weeks of live. An important issue in this field is of course the cost of such a treatment, which to a high degree depends on the cost of producing the immunoglobulin concentrate.
Isolates of animal immunoglobulins, e.g. from bovine milk, are also under investigation as an oral health care or even therapeutic product to avoid or treat gastrointestinal infections, e.g. in AIDS patients. For such applications both the degree of purity of the product as well as the cost is of major importance.
A more sophisticated application of antibodies for therapeutic use is based on so called “drug-targeting” where very potent drugs are covalently linked to antibodies with specific binding affinities towards specific cells in the human organism, e.g. cancer cells. This technique ensures that the drug is concentrated on the diseased cells giving maximal effect of the drug without the severe side-effects that frequently occurs when using chemotherapy. For such purposes the antibodies have to be very carefully controlled and of high purity, and the typical way of performing the primary production are either by producing monoclonal antibodies in hybridoma cell culture or by fermenting genetically engineered bacteria, e.g.
E.coli
Isolation of Immunoglobulins
All the above mentioned applications of immunoglobulins requires some sort of isolation of the antibody from the crude raw material, but each kind of application has its own very varying demands with respect to the final purity and allowable cost of the antibody product
Generally, there exists a very broad range of different methods available for isolation of immunoglobulins giving a very broad range of final purities, yields and cost of the product.
Traditional methods for isolation of immunoglobulins are based on selective reversible precipitation of the protein fraction comprising the immunoglobulins while leaving other groups of proteins in solution. Typical precipitation agents being ethanol, polyethylene glycol, lyotropic (anti-chaotropic) salts such as ammonium sulfate and potassium phosphate, and caprylic acid.
Typically, these precipitation methods are giving very impure products while at the same time being time consuming and laborious. Furthermore, the addition of the precipitating agent to the raw material makes it difficult to use the supernatant for other purposes and creates a disposal problem. This is particularly relevant when speaking of large scale purification of immunoglobulins from, e.g., whey and plasma.
Ion exchange chromatography is another well known method of protein fractionation frequently used for isolation of immunoglobulins. However, this method is not generally applicable because of the restraints in ionic strength and pH necessary to ensure efficient binding of the antibody together with the varying isoelectric points of different immunoglobulins.
Protein A and Protein G affinity chromatography are very popular and widespread methods for isolation and purification of immunoglobulins, particularly for isolation of monoclonal antibodies, mainly due to the ease of use and the high purity obtained. Although being popular it is however recognised that Protein A and Protein G poses several problems to the user among which are: very high cost, variable binding efficiency of different monoclonal antibodies (particularly mouse IgG
1
), leakage of Protein A/Protein G into the product, and low stability of the matrix in typical cleaning solutions, e.g. 1 M sodium hydroxide. Each of these drawbacks have its specific consequence in the individual application, ranging from insignificant to very serious and prohibitive consequences.
Hydrophobic chromatography is also a method widely described for isolation of immunoglobulins, e.g in “Application Note 210, BioProcess Media” published by Pharmacia LKB Biotechnology, 1991. In this reference a state of the art product “Phenyl Sepharose High Performance” is described for the purpose of purifying monoclonal antibodies from cell culture supernatants. As with other hydrophobic matrices employed so far it is necessary to add lyotropic salts to the raw material to make the immunoglobulin bind efficiently. The bound antibody is released from the matrix by lowering the concentration of lyotropic salt in a continuous or stepwise gradient. It is recommended to combine the hydrophobic chromatography with a further step if highly pure product is the object.
The disadvantage of this procedure is the necessity to add lyotropic salt to the raw material as this gives a d problem and thereby increased cost to the large scale user. For other raw materials than cell culture supernatants such as whey, plasma, and egg yolk the addition of lyotropic salts to the raw materials would in many instances be prohibitive in large scale applications as the salt would prevent any economically feasible use of the immunoglobulin depleted raw material in combination with the problem of disposing several thousand liters of waste.
Thiophilic adsorption chromatography was introduced by J. Porath in 1985 (J. Porath et al; FEBS Letters, vol. 185, p.306, 1985) as a new chromatographic adsorption principle for isolation of immunoglobulins. In this paper, it is described how divinyl sulfone activated agarose coupled with various ligands comprising a free mercapto-group show specific binding of immunoglobulins in the presence of 0.5 M potassium sulfate, i.e. a lyotropic salt. It was postulated that the sulfone group, from the vinyl sulfone spacer, and the resulting thio-ether in the ligand was a structural necessity to obtain the described specificity and capacity for binding of antibodies. It was however later shown that the thio-ether could be replaced by nitrogen or oxygen if the ligand further comprised an aromatic radical (K. L. Knudsen et al, Analytical Biochemistry, vo
Hansen Marie Bendix
Lihme Allan Otto Fog
Russel Jeffrey E.
Upfront Chromatography A/S
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