Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1997-08-21
2001-04-03
Saunders, David (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S380000, C530S381000, C530S383000, C530S384000, C424S530000
Reexamination Certificate
active
06211344
ABSTRACT:
The invention relates to a process for preparing deficient plasmas, in particular a factor V-deficient plasma, from a starting plasma using antibodies.
A factor V-deficient plasma is understood as being a plasma which is suitable for blood coagulation investigations and is free of factor V but which contains, at essentially normal concentration, all the other coagulation factors which are normally present in the plasma.
A factor V-deficient plasma is suitable, in combination with suitable coagulation tests, for determining the content of activatable or inactivatable factor V in factor V-containing fluids, for example blood or plasma.
The principle of such a determination consists in maxing the sample to be determined with an excess of factor V-deficient plasma and carrying out a coagulation test of known type. The result of this coagulation test then depends only on the content of factor V which is present in the blood sample or plasma sample under investigation, since all the remaining coagulation factors are present in excess and consequently not rate-limiting for the reaction.
In order to determine activatable factor V, a thromboplastin time (TPT), for example, is determined for the sample mixture. In this case, a coagulation activator, i.e. thromboplastin in the case of the TPT, phospholipids and calcium chloride are added to the patient sample which has been mixed with the deficient plasma and the coagulation time is determined. In the absence of factor V, the coagulation of the mixture is slowed down markedly, while in the presence of factor V the coagulation time is shortened in dependence on the factor V activity which is present. This method has been described, for example, by H. Stormorken (The preparation of proaccelerin deficient (parahemophilia) plasma for the assay of proaccelerin. Scand J Clin Lab Invest 9: 273, 1957).
In order to determine inactivatable factor V, an activated partial thromboplastin time (APTT), for example, is carried out on the sample mixture in the presence of activated protein C (see, for example: Kraus, M., Zander, N. and Fickenscher, K.: Coagulation assay with Improved specificity to factor V mutants insensitive to activated protein C. Thrombosis Research, 80: 255-264, 1995; and EP 0 711 838). In the presence of a normally inactivatable factor V, the coagulation time in the presence of activated protein C is prolonged as compared with the APTT. However, if factor V cannot be inactivated by activated protein C, the prolongation is then less pronounced.
The sensitivity of the determination of activatable factor V, in particular, depends crucially on the quality of the deficient plasma employed with regard to the residual content of factor V in the deficient plasma, since the determination is carried out using an excess of deficient plasma and, as a consequence, even a small proportion of factor V, for example 1% residual activity, can no longer be tolerated.
When inactivatable factor V is determined, discrete values are to be expected because this determination is carried out for the purpose of detecting a genetic defect which is also termed factor V disorder. Thus, in a heterozygous carrier of inactivatable factor V, the prolongation of the coagulation time is approx. 50%, while in a homozygous carrier it is close to 0%. On the other hand, the determination of activatable factor V is directed, in particular, to the detection of acquired deficiencies which appear, for example, during operations or in association with liver damage, so that values for activatable factor V can be expected within the entire concentration range. The detection of very low concentration of activatable factor V, for example within a range of between 1 and 10%, is particularly important for diagnosis. The risk of patients with a factor V deficiency suffering uncontrolled internal bleeding increases very markedly below 10% factor V activity. A particularly exact determination is therefore required in this range in order to decide on a therapy, for example using fresh plasma. If the residual activity of the factor V is in the range from about 1 to 5% in a deficient plasma which is supplied in excess, the coagulation test becomes insensitive, i.e. the reference curve becomes too flat, with the result that reliable measurements cannot be carried out in the critical, low concentration range between about 1 and 10%.
A useful deficient plasma should therefore have a factor V activity which is markedly less than 1% of the normal activity.
A deficient plasma of the quality which the above account indicates is imperative can be the plasma of patients who themselves have factor V activities which are markedly less than 1%. A deficient plasma of this type is naturally rare and is not available in sufficient quantity for routine purposes, and ethical problems are attached to obtaining blood donations from patients who are suffering from this severe form of hemophilia.
It is therefore necessary to look for other ways of preparing a suitable deficient plasma. In this context, the possibility suggests itself of destroying the factor V by physicochemical means, with, for example, factor V being denatured, and thereby inactivated, by adding EDTA (ethylenedinitrilotetraacetic acid) and heating. The method of Janssen et al. (Janssen, C. L., Wijngaards, G. and van der Meer, J.: Conditions for stabilization and determination of activated factor V. Thrombosis Research 5: 315-325, 1974) represents an example of this approach. However, this deficient plasma is not suitable for determining inactivatable factor V since factor VIII is also destroyed by adding EDTA (see EP 0 711 838).
Another known method consists in removing factors immuno-adsorptively by means of antibodies. Specific antibodies are bound to an insoluble support material, and the plasma is brought into contact with this support material, to which the specific antibody is bound, so that the relevant factor is removed from the plasma, thereby making it possible to prepare a deficient plasma. In contrast with the above-described chemical methods, other factors, in particular factor VIII, which is important for determining inactivatable factor V, are not destroyed. For this reason, it is only deficient plasmas which have been prepared in this way which can be used for determining inactivatable and activatable factor V (see EP 0 711 838; Kraus et al. 1995).
However, factor V-deficient plasmas which have hitherto been prepared by the immunoadsorptive method are markedly inferior to the chemical method in their sensitivity with regard to activatable factor V. This is demonstrated by way of example in Example 1, in which reference curves for determining activatable factor were constructed using chemically prepared and immunoadsorptively prepared factor V-deficient plasmas, respectively. In the case of the plasma which was purified immunoadsorptively in accordance with the state of the art, the spread of the reference curve in the critical region between factor V contents of 1% and 10% is only about 40% of the spread which was obtained using the chemically prepared plasma.
The use of a combination of antibodies against two different antigens has also been described already, for example in EP 0 281 089 for the purpose of preparing a factor VIII-deficient plasma. Since factor VIII:C is bound to von Willebrand factor in the plasma, an appropriately high quality, i.e. low concentrations of factor VIII:C, was only achieved using a combination of an immunoadsorptive purification against von Willebrand factor and then an immunoadsorptive purification against factor VIII:C. However, in this method, the sequence of the purification steps is important as is the fact that the antibodies employed are of high specificity. By contrast, the sequence of the purification steps is of no significance in the process according to the invention and antibodies are used twice against the same antigen.
The invention was therefore based on the object of developing a process for preparing a factor V-deficient plasma, which process exhibits high sensitivity, whic
Aillaud Erika
Drescher Heinz-Hermann
Kraus Michael
Dade Behring Marburg GmbH
De Cloux Amy
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Saunders David
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