Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Recovery or purification
Reexamination Certificate
2000-02-23
2002-06-04
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Recovery or purification
C422S001000, C422S105000, C210S641000, C210S644000, C210S645000, C210S649000, C210S650000, C210S651000, C210S653000, C210S767000
Reexamination Certificate
active
06399357
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method of virus-filtering a solution that contains at least one macromolecule, by virtue of the total salt content of the solution lying in the range of from about 0.2 M up to saturation of the solution with the salt concerned. The inventive method reduces the residence time and the extent to which the solution need to be diluted, and optimizes the yield when virus-filtering primarily proteins, polysaccharides and polypeptides. The reduction in virus content is at least as good as with conventional techniques where the total salt content is low. The present invention facilitates virus filtration with the aid of the so-called “dead-end” technique, which affords several process and economic advantages in comparison with the tangential virus-filtering technique normally used. When virus-filtering the plasma protein factor IX, the yield obtained in the virus-filtering stage is increased from about 70% to above 95%, by raising the salt content of the solution in accordance with the present invention.
BACKGROUND OF THE INVENTION
The problem of virus contamination of various protein preparations intended for the medication of human beings has received greater notice in recent years. For instance, occasional reports have been submitted concerning, e.g., blood proteins that have been contaminated with hepatitis virus A, hepatitis virus B, hepatitis virus C and/or Human Immunodeficiency Virus (HIV). In keeping with these reports, the authorities of several countries have sharpened their requirements with regard to cleansing protein preparations of their possible virus contaminants.
In present-day, conventional techniques, viruses are inactivated with the aid of chemical additives, primarily solvents and detergents, and/or by exposing the viruses to elevated temperatures. The former method has the drawback of functioning solely on virus with lipid envelopes, for instance hepatitis virus B and HIV. The latter technique mentioned above has the drawback that many proteins are thermally unstable at those temperatures required to effectively reduce the contaminating virus.
U.S. Pat. No. 4,473,494 (assigned to the U.S. Secretary of the Army) discloses a method for production of stroma-free, non-heme protein-free hemoglobin by use of zinc ions to promote precipitation of a zinc ion-bound insoluble hemoglobin complex, followed by membrane ultrafiltration of the zinc-hemoglobin complex from the filtrate fluid medium. In the only step where viruses are said to be removed from hemoglobin, the total salt content is below 0.05 M, i.e. the total content of salt is conventional.
EP-A-0307373 (assigned to Ares-Serono) relates to removal of viruses and/or other contaminants from biological materials in fluid form by using ultrafiltration membranes having a 100,000 Da cut-off. A preferred biological material is human growth hormone. In the examples of EP-A-0307373, the total content of salt in the virus-filtering step lies in the range of from 0.01 up to 0.10 M (NH
4
CO
3
), i.e. the total content of salt is conventional.
There is thus a need for an effective virus-reducing method which can be applied to different types of macromolecules, primarily proteins, and on different types of viruses.
DESCRIPTION OF THE INVENTION
One object of the present invention is to markedly reduce the residence time when virus-filtering solutions that contain macromolecules.
Another object of the present invention is to markedly reduce the liquid volumes when virus-filtering solutions that contain macromolecules.
A further object of the present invention is to reduce the filter area required to effectively virus-filter solutions that contain macromolecules.
Yet another object of the present invention is to achieve a macromolecule yield in excess of about 90% in the virus-filtering stage.
Still another object of the present invention is to reduce the polymerization obtained on the virus filter surface, so as to enable the rate of flow to be increased and the process time to be decreased.
These and other objects are fulfilled by the present invention, which relates to a method of virus-filtering a solution containing at least one macromolecule wherein the total salt content of the solution lies within the range of from about 0.2 M up to saturation of the solution with the salt concerned.
The inventor of this invention has thus found that virus filtration can be effected much more effectively than previously known, by increasing the salt content of the solution. This discovery is surprising, because hitherto in virus filtration of proteins it has been believed that solely the protein concentration, the rate of flow and the pH have had any influence on the process.
It is believed that the enhanced filtering effect achieved at higher salt concentrations is because the protein contracts and can therewith pass more easily through the filter pores. It is also conceivable, that the interaction is reduced between macromolecules themselves and/or between the macromolecules and the material of the filter membrane. It is also conceivable that proteins having a large number of hydrophobic groups are influenced to a greater extent by an elevated salt concentration.
The closer the molecular weight, or relative molecular mass, of the macromolecule lies to the pore size of the filter membrane, the more effective the present invention. The effectiveness of the present invention is also enhanced when the difference in the size and/or the molecular weight of the contaminants and the product increases, i.e. with increasing concentrations of high molecular contaminants in the product.
The present invention also facilitates specific fractions to be separated from a desired product, for instance enables undesirable proteins to be separated from the protein that constitutes the product.
The use of a high salt content according to the present invention, also enables the use of the socalled “dead-end” filtering technique. This preferred embodiment, has several advantages over conventional tangential filtering processes normally applied, especially with a pore size of about 5-30 nm. For instance, the equipment and operating procedures required are much simpler and therewith less expensive. The use of “dead-end” filtration also reduces the loss of the macromolecule, reduces the process time, increases the permeability of the macromolecule through the filter, and also enables a generally constant concentration of the macromolecule to be achieved over the filter as well as a constant membrane pressure. Another advantage with the dead-end filtering technique, is the fact that scaling-up of virus filtering processes from laboratory to industrial scale is considerably facilitated.
When practicing the present invention, the total salt content of the solution suitably lies within the range of from 0.3 up to 3.0 M, preferably within the range of from 0.4 up to 2.5 M, and more preferably within the range of from 0.6 up to 2.0 M. It is particularly preferred that the total salt content of the solution lies within the range of from 0.8 up to 1.5M.
When necessary, the total salt content of the solution can be adjusted by adding any acceptable salt. For instance, it is possible to use soluble inorganic salts, soluble organic salts or combinations of such salts. It is assumed that important process advantages are obtained when using salts which exhibit a high salting-out effect in accordance with the so-called Hofmeister series. Reference is here made to S. Glasstone, Textbook of Physical Chemistry, van Nostrand Co., Toronto, 2
nd
edition, April 1946, pp. 1254-1259. The most important examples of anions which have such high salting-out effect are citrate, tartrate, sulfate, acetate and phosphate. Cations that can be used advantageously when practicing the present invention are monovalent cations, such as sodium, potassium and ammonium, as well as divalent cations, such as calcium. Sodium chloride, potassium chloride, sodium acetate and sodium citrate or combinations thereof are particularly preferred salts in accord
Biovitrum AB
Fish & Richardson P.C.
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