JPS61155332A - Method of treating blood coagulation factor viii - Google Patents

Abstract

PURPOSE:To treat the titled factor, by lyophilizing purified and concentrated blood cogulation factor VIII, and heating it at a specific temperature for a specified time. CONSTITUTION:Purified and concentrated blood coagulation factor VIII is lyophilized, heat-treated at 60-65 deg.C for preferably 72-96hr, to give stable pharmaceutical preparation of blood coagulation factor VIII(F VIII). F VIII is one of pharmaceutical preparations of plasma fraction, and is essential to hemostasis of a patient of hemophilia A. In pool plasma of raw material, contam ination with various bacteria or viruses, especially contamination with hepatitis is a big problem, and the heat treatment sufficiently deactivates not only hepati tis virus but also other viruses and is a treatment condition for stabilizing F VIII.

Description

[Detailed description of the invention] The present invention relates to a method for treating blood coagulation factor (I).

Blood coagulation factor ■ preparation (hereinafter referred to as F■) is one of the plasma fraction preparations and is essential for hemostasis in hemophilia A patients. This F■ is fractionated, concentrated and purified from plasma, and its raw material is usually derived from pooled plasma from several thousand suppliers.

Therefore, contamination by various microorganisms or viruses, especially by hepatitis virus, has become a major problem.

Heat treatment is effective for removing such viral contamination, that is, inactivating viruses, especially heat-stable plasma fraction preparations.
For example, it has been clinically established that albumin preparations can be treated in solution form at 60° C. for 110 hours to sufficiently prevent virus propagation. However, F■ is unstable to heat in a solution state, and under the above heat treatment conditions, the F■ activity itself is inevitably reduced significantly. For this reason, when treating solutions containing F■, various stabilizers such as amino acids such as glycine, chlorosaccharides of glucose, chlorosaccharides of mannitol, etc., or combinations thereof may be added. is being attempted. However, in the presence of such a stabilizer, the virus is also stabilized at the same time, and the current situation is that a sufficient effect of inactivating the virus cannot be expected.

Recently, a so-called dry heating method has been proposed for processing plasma fraction preparations containing F■, in which these preparations are subjected to heat treatment in a freeze-dried state (Japanese Unexamined Patent Publication No. 58-213721). The main point of this method is to treat each formulation in a freeze-dried state at a temperature of 60°C or higher, and the F
A treatment method for up to about 100'C127 hours is disclosed. According to this, the decrease in coagulation activity is within an acceptable range;
It is said that it is sufficient to inactivate various viruses such as hepatitis virus, EB virus, etc.

However, there is no mention of whether coagulation activity is maintained by longer treatment or whether viruses that are more resistant to heat, such as parvovirus, are also inactivated.

Rather, according to the experiments of the present inventors, dry heating of F
It has been found that non-A and non-B hepatitis viruses are not sufficiently inactivated at a temperature of 30 hours, and that coagulation activity is significantly reduced at temperatures of 70° C. or higher.

The present invention is based on the basic experiments of the present inventors, and in dry heating of F■, the heating temperature is 60 to 6
The present invention was completed based on the discovery that 72 to 96 hours is desirable as the heating time at 5°C.

The experimental results of the present inventors will be explained below.

First, the effect of heating on the activity of F■ was investigated by varying the temperature. In Figure 1, the freeze-dried F preparation was heated at 60, 65, 70, 80, 90, and 100°C.
(2) Activity was measured by the Hardj-Sty one-step method.

This experiment investigated the decrease in activity of F■ under extremely long-term heating.If the heating temperature was 65℃, the decrease in activity was within 20% even after long-term heating for 15 days, indicating excellent stability. I came to know that Naturally, the higher the heating temperature, the faster the decrease in F■ activity. Therefore, from the standpoint of stability of F■ activity, it is known that it is desirable to keep the temperature at 65° C. or lower and to extend the heating time.

Next, FIG. 2 shows inactivation songs Pa by heating of various viruses used in experiments such as picornavirus and orthomyxovirus. These viruses were added to F■ and freeze-dried. The freeze-dried product was subjected to heat treatment at two levels: 6o0C and 65°C, and the amount of virus was measured at each time. The virus infectivity titer was expressed as so or PFU from TC. In the figure, the amount of virus in the Heha undiluted solution, the mouth is the mixture of virus and F■, and the symbol ◎ is the amount of virus in the sample after freeze-drying.・ indicates the amount of virus when heat treated at 600C, and ○ indicates the amount of virus when heat treated at 65℃. Group 1 viruses can be inactivated only by freeze-drying, so there is no need for heating. Polio, x
Echo, Coxsack4
e) and other picornaviruses. Next, the second group is measles and mumps.
It is known that these types of viruses are relatively heat-resistant, as the Orthomyxo viruses of influenza A and B are inactivated by heating for 5 to 10 hours.

The viruses shown in the third group are heat-resistant, and the inactivation status of virus titer versus heating time is shown in FIG. Herpes simplex virus (I (S■)) is inactivated by heat treatment at 60°C for about 10 hours, but rubella (Rubella) and Japanese encephalitis (JEV) Toga virus are relatively heat-resistant and A heat treatment of 30 to 30 hours is required. Varicella (VZ) in particular is quite resistant to heat and requires a heat treatment of 35 hours at 60°C or 20 hours at 65°C. Viruses that are sensitive to heat are Although there is almost no difference in treatment time between heating at 60°C and 65°C, in tests for viruses that are relatively heat resistant, a slight difference in heating time between 60°C and 65°C is observed.

Porcine parvo (Porci) is a heat-resistant virus.
Fig. 4 shows an inactivation test by heating conducted for virus f.

The upper curve in Figure 4 shows the result obtained by adding TCIDso's Balbowi Res-4h''■ and freeze-drying and drying and heating at 65℃ to 71%.In reality, such a high titer virus It was confirmed that even those with an extremely high 1 vacancy value of 10 (lower curve in Figure 4) can be completely inactivated by heating at 65°C for 196 hours. These results also indicate that even when heated at 65°C for 196 hours, It is reported that one case in each city is enough to eliminate the virus for the first time in a row.

Next, a comparative test was conducted between drying heat of F■ and heating of albumin J for 2 nights. 2ON, /'V% albumin f81
Figure 5 shows a comparison of the virus inactivation curves of the product that was heated at 600C for two nights with Takuparvovirus added thereto, and the one that was dried and heated at 65°C using the F formulation that also added Takuparvovirus. be.

As shown in Figure 5-1, virus inactivation progresses more rapidly when heated at 60°C for two nights than when dried at 65°C after freeze-drying. When this relationship is illustrated in FIG. 5-2 by changing the time scale, it can be seen that the liquid heating of the albumin solution at 60°C for 10 hours corresponds to the freeze-drying and heating of F2 at 65°C for 172 hours. Therefore, as mentioned at the beginning, 60% is known to be a condition that guarantees the inactivation of hepatitis viruses in albumin preparations.
It is known that dry heating at 65°C for 172 hours is required in F2 compared to overnight heating at 65°C for 110 hours.

Based on the above results, the present inventors found that the freeze-drying heating method is superior in stabilizing F■, and that even some viruses that are extremely heat-resistant can be stabilized at a temperature of 65°C.
Although it was confirmed that the virus could be inactivated by heating for 6 hours, the effect of heating on hepatitis viruses that may actually be mixed into plasma components was investigated through animal experiments. In particular, in viv for N0nA-nonB hepatitis, which is said to be impossible to clean even if it is currently contaminated.
o? A strike was carried out.

Serum from a chimpanzee that developed N0nA-nonB hepatitis (donated by Professor Shikata, Nihon University School of Medicine) was collected from F■
After adding to the formulation, lyophilize and prepare NonA-nonB
We created an FVI preparation that is thought to contain type hepatitis virus. The preparation was divided into three groups, and an inoculation experiment was conducted on chimpanzees using one without heat treatment, one with heat treatment at 60°C for 172 hours, and one with heat treatment at 65°C for 196 hours. According to control Vi 13 weeks later, GOT, GPT, -GT
An increase in P was observed.

The samples that were heat-treated at 60°C for 172 hours were inoculated into two chimpanzees. In one of these animals, increases in GOT, GPT, and -GTP were observed as in the control. No change was observed in one of the animals in the pond, but a slight increase in -GTP was observed. No signs of hepatitis were observed in the two chimpanzees that received the samples heat-treated at 65°C for 196 hours.

As a result, it was confirmed that dry heating at 65° C. for 196 hours is desirable as a condition under which the virus inactivation effect can be expected without reducing the activity of F■. The properties of FMI preparations heated under these conditions include physicochemical properties, safety tests in rats, mice, and dogs, the chimpanzee test described above, solubility, E(PLO, ultraviolet absorption, fluorescence, CD spectrum, etc.). No change was observed before and after the heat treatment.Thus, the heat treatment method according to the present invention is sufficient to inactivate only the hepatitis virus and the oil virus, and the treatment conditions also contribute to the stabilization of KariF. be.

[Brief explanation of drawings]

FIG. 1 is a graph showing changes in F■ activity due to dry heating. FIGS. 2 and 3 show inactivation curves showing the time-dependent changes in virus titer due to freeze-drying and heating of each group of viruses. Figure 4 shows the inactivation curve of porcine parvovirus by heating.
Figure 5 shows the inactivation of porcine parvovirus by heating at 60°C in 20% albumin, and F
This is a graph comparing those obtained by drying and heating at 65°C.

Claims (1)

[Claims] 1. A method for producing blood coagulation factor VIII, which is characterized in that purified and concentrated blood coagulation factor VIII is freeze-dried and then heated at a heating temperature of 60°C to 65°C for at least 72 hours. Processing method. 2. The method according to item 1 above, wherein the method is heated at a heating temperature of 65° C. for 96 hours or more.