News Column

Patent Application Titled "Method for Manufacturing Protein Drug" Published Online

August 5, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- According to news reporting originating from Washington, D.C., by NewsRx journalists, a patent application by the inventors Hongo, Tomoko (Chiyoda-ku, JP); Hayashida, Hirohisa (Chiyoda-ku, JP), filed on June 22, 2012, was made available online on July 24, 2014 (see also Asahi Kasei Medical Co., Ltd.).

The assignee for this patent application is Asahi Kasei Medical Co., Ltd.

Reporters obtained the following quote from the background information supplied by the inventors: "Protein drugs typified by biomedicines, plasma derivatives, and the like have raised concerns about contamination by ingredient-derived or process-derived viruses. Thus, when such protein drugs are manufactured, the inactivation or removal of viruses in the drugs is very important from the viewpoint of the safety and stability of the drugs. This inactivation of viruses has been practiced by a method such as heat treatment or treatment with chemical agents. These treatments, however, are not sufficient in themselves for the inactivation of viruses. In addition, these methods might denature the proteins themselves in the drugs. Against this backdrop, the viruses are separated and removed by filtration using virus removal membranes as physical virus removal means without chemical denaturation (e.g., Patent Literatures 1 to 3).

"Virus removal membranes made of natural materials such as cellulose or of synthetic polymer materials such as polyvinylidene fluoride (PVDF) or polyether sulfone (PES) are known (Non Patent Literatures 1 to 4). Particularly, in the case of protein solutions containing small protein molecules, small-pore size virus removal membranes having a pore size that does not permit permeation of viruses but permits permeation of the protein molecules are used.

"Ideally, the filtration of virus-containing solutions using a virus removal apparatus equipped with a virus removal membrane should filter larger amounts of protein solutions in a short time and should exert sufficiently high virus removal performance. To treat larger amounts of protein solutions in a short time, filtration of virus-containing solutions is generally carried out at a pressure as high as possible. However, the continuation of such high-pressure filtration may leave inside the membrane, proteins supposed to be contained in filtrates. In addition, recent protein drugs tend to have higher concentrations of proteins. Along with this tendency, there is also a growing demand for higher protein concentrations in the filtration step for removing viruses. In the case of filtering high-concentration protein solutions through a small-pore size virus removal membrane, clogging frequently occurs, particularly, due to proteins remaining inside the membrane.

"Such proteins remaining inside the small-pore size virus removal membrane are recovered by filtration with a protein-free buffer solution (usually, the same as a buffer solution used for dissolving the proteins) as a washing solution. This filtration step is added after the protein filtration and therefore called post-wash or post-filtration. For this post-wash, typically, the filtration pressure is temporarily relieved in order to have a switching, at an entrance of solution to be filtered, from a line for protein solutions to a line for washing solutions. If the filtration pressure is not decreased, the solution flows backward to the washing solution side.

"Examples of such decrease in filtration pressure during filtration through a virus removal membrane, as in the post-wash step, include a case in which pressurization is suspended during filtration for a reason such as a power failure (this case is called stop and start).

"Depending on the types of protein drugs, low filtration pressures may be desirable for filtration through a virus removal membrane during manufacturing of the drugs. Such filtration at low filtration pressures is often carried out in order to increase the final throughputs of solutions that tend to cause clogging or in order to increase the rate of permeation or recovery of solutions of high-molecular proteins in elongated shapes. When low filtration pressures are adopted, specific filtration pressures are often determined to balance permeability and productivity and also depend on the concentrations, etc. of the protein drugs to be obtained. For example, Patent Literature 4 has adopted a filtration pressure on the order of 0.15 kgf/cm.sup.2."

In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventors' summary information for this patent application: "Technical Problem

"In the conventional filtration of protein solutions using virus removal membranes, attentions have been focused on methods performed at a pressure as high as possible in order to increase throughputs and enhance efficiency. Sufficient findings have not been gained about filtration at low filtration pressures.

"Against this backdrop, the present inventors have conducted their own studies on the filtration of protein solutions using small-pore size virus removal membranes at low filtration pressures and, surprisingly, have found that when filtration at low filtration pressures is carried out under solution conditions similar to those at high filtration pressures, viruses may be leaked into filtrates, depending on the solution conditions, resulting in protein drugs having low rates of virus removal. The present inventors have also found that filtration in the post-wash step or the stop and start step, which is also performed at low filtration pressures, may therefore have a reduced rate of virus removal, depending on the solution conditions.

"On the basis of these novel findings, an object of the present invention is to provide a method for manufacturing a virus-free protein drug, comprising a step of filtering a virus-containing protein solution through a small-pore size virus removal membrane at a low filtration pressure, wherein the rate of virus removal by the method for manufacturing the virus-free protein is high.

"Solution to Problem

"The present inventors have conducted diligent studies to attain the object and consequently completed the present invention by finding that a protein drug having a high rate of virus removal can be obtained even at a low filtration pressure by setting the pH and salt ionic strength of a solution to be filtered to particular values.

"Specifically, the present invention relates to the followings:

"[1]

"A method for manufacturing a virus-free protein drug, comprising the following step (a):

"(a) a filtration step of filtering a virus-containing protein solution through a small-pore size virus removal membrane to obtain a virus-free protein solution,

"the filtration step (a) comprising the following step (q):

"(q) a low-pressure filtration step of filtering the solution through the small-pore size virus removal membrane at a filtration pressure of 0.30 kgf/cm.sup.2 or lower to obtain the virus-free protein solution,

"wherein the solution prior to filtration in the low-pressure filtration step (q) has a pH (X) and a salt ionic strength (Y (mM)) that satisfy the following equations 1 and 5:

"0.ltoreq.Y.ltoreq.150X-590 (Equation 1)

"3.5.ltoreq.X.ltoreq.8.0 (Equation 5)

"or the following equations 4 and 5:

"Y=0 (Equation 4)

"3.5.ltoreq.X.ltoreq.8.0 (Equation 5).

"[2]

"The method according to [1], wherein the solution prior to filtration in the step (q) is the virus-containing protein solution, and

"wherein 50% or more of the whole virus-containing protein solution to be filtered in the filtration step (a) is filtered in the low-pressure filtration step (q).

"[3]

"The method according to [1], wherein the filtration step (a) is a step of filtering the virus-containing protein solution through the small-pore size virus removal membrane at a filtration pressure of 0.30 kgf/cm.sup.2 or lower to obtain the virus-free protein solution, wherein the solution prior to filtration in the filtration step (a) has a pH (X) and a salt ionic strength (Y (mM)) that satisfy the following equations 1 and 5:

"0.ltoreq.Y.ltoreq.150X-590 (Equation 1)

"3.5.ltoreq.X.ltoreq.8.0 (Equation 5)

"or the following equations 4 and 5:

"Y=0 (Equation 4)

"3.5.ltoreq.X.ltoreq.8.0 (Equation 5).

"[4]

"The method according to [1], wherein the filtration step (a) comprises the following step (p) performed prior to the low-pressure filtration step (q): (p) a high-pressure filtration step of filtering the virus-containing protein solution through the small-pore size virus removal membrane at a filtration pressure exceeding 0.30 kgf/cm.sup.2 to obtain the virus-free protein solution.

"[5]

"The method according to [4], wherein the solution prior to filtration in the low-pressure filtration step (q) is a buffer solution for washing.

"[6]

"The method according to [4] or [5], wherein the low-pressure filtration step (q) is a post-wash step or a stop and start step.

"[7]

"The method according to any of [1] to [6], wherein the filtration solution in the low-pressure filtration step (q) has a pH (X) and a salt ionic strength (Y (mM)) that satisfy the following equations 2 and 5:

"0.ltoreq.Y.ltoreq.50X-200 (Equation 2)

"3.5.ltoreq.X.ltoreq.8.0 (Equation 5)

"or the following equations 4 and 5:

"Y=0 (Equation 4)

"3.5.ltoreq.X.ltoreq.8.0 (Equation 5).

"[8]

"The method according to any of [1] to [6], wherein the solution prior to filtration in the low-pressure filtration step (q) has a pH (X) and a salt ionic strength (Y (mM)) that satisfy the following equations 3 and 5:

"0.ltoreq.Y.ltoreq.50X-250 (Equation 3)

"3.5.ltoreq.X.ltoreq.8.0 (Equation 5)

"or the following equations 4 and 5:

"Y=0 (Equation 4)

"3.5.ltoreq.X.ltoreq.8.0 (Equation 5).

"[9]

"The method according to any of [1] to [8], wherein the low-pressure filtration step (q) is a step of filtering the solution through the small-pore size virus removal membrane at a filtration pressure of 0.20 kgf/cm.sup.2 or lower to obtain the virus-free protein solution.

"[10]

"The method according to any of [1] to [4], wherein a log reduction value (LRV) calculated according to the following equation 6 is 4 or higher:

"LRV=log.sub.10(C.sub.0/C.sub.F) (Equation 6)

"wherein C.sub.0 represents the virus concentration of the virus-containing protein solution before the filtration step (a), and C.sub.F represents the virus concentration of the virus-free protein solution after the filtration.

"[11]

"The method according to [5] or [6], wherein a log reduction value (LRV) calculated according to the following equation 6 is 4 or higher:

"LRV=log.sub.10(C.sub.0/C.sub.F) (Equation 6)

"wherein C.sub.0 represents the virus concentration of the virus-containing protein solution before the filtration step (a), and C.sub.F represents the virus concentration of the virus-free protein solution after the filtration, and LRV' calculated according to the following equation 7 is 4 or higher:

"LRV'=log.sub.10(C.sub.0/C.sub.w) (Equation 7)

"wherein C.sub.0 represents the virus concentration of the virus-containing protein solution before the filtration step (a), and C.sub.w represents the virus concentration of the filtrate of the buffer solution for washing after the filtration step (a). [12]

"The method according to any of [1] to [11], wherein the material of the small-pore size virus removal membrane is cellulose or a hydrophilized synthetic polymer.

"[13]

"The method according to any of [1] to [12], wherein the material of the small-pore size virus removal membrane is a hydrophilized synthetic polymer, and wherein the synthetic polymer is selected from the group consisting of polyvinylidene fluoride, polyether sulfone, polysulfone, and polyethylene.

"[14]

"The method according to any of [1] to [13], wherein the form of the small-pore size virus removal membrane is a flat membrane or a hollow fiber membrane.

"[15]

"The method according to any of [1] to [14], wherein the virus-containing protein solution has a protein concentration of 1 mg/mL to 100 mg/mL.

"[16]

"The method according to any of [1] to [15], wherein the virus-containing protein solution comprises one or more protein(s) selected from the group consisting of various monoclonal antibodies, recombinant blood coagulation factor, interferon, various hormones, various enzymes, immunoglobulin, albumin, blood coagulation factor VIII, blood coagulation factor IX, fibrinogen, and antithrombin III.

"[17]

"The method according to any of [1] to [15], wherein the virus-containing protein solution comprises an antibody as the protein.

"[18]

"The method according to any of [1] to [15], wherein the virus-containing protein solution comprises blood coagulation factor VIII or fibrinogen as the protein.

"[19]

"The method according to any of [1] to [18], wherein the virus-containing protein solution comprises one or more virus(es) selected from the group consisting of human parvovirus B19 (B19), minute virus of mice (MVM), porcine parvovirus (PPV), bovine parvovirus (BPV), canine parvovirus (CPV), poliovirus (Polio), circovirus, hepatitis A virus (HAV), and hepatitis E virus (HEV).

"[20]

"The method according to any of [1] to [19], wherein the virus-containing protein solution comprises a virus of 32 nm or smaller in diameter having no envelope.

"[21]

"The method according to any of [1] to [20], wherein the virus-containing protein solution comprises one or more component(s) selected from the group consisting of an inorganic salt, a buffer solution component, a surfactant, and a saccharide.

"[22]

"A method for manufacturing a virus-free protein drug, comprising the following step (a):

"(a) a filtration step of filtering a virus-containing protein solution through a small-pore size virus removal membrane to obtain a virus-free protein solution,

"the filtration step (a) comprising the following step (q):

"(q) a low-pressure filtration step of filtering the solution through the small-pore size virus removal membrane at a filtration pressure of 0.30 kgf/cm.sup.2 or lower to obtain the virus-free protein solution,

"and comprising, prior to the low-pressure filtration step (q), a step of adjusting the solution prior to filtration so that the solution prior to filtration in the step (q) has a pH (X) and a salt ionic strength (Y (mM)) that satisfy the following equations 1 and 5:

"0.ltoreq.Y.ltoreq.150X-590 (Equation 1)

"3.5.ltoreq.X.ltoreq.8.0 (Equation 5)

"or the following equations 4 and 5:

"Y=0 (Equation 4)

"3.5.ltoreq.X.ltoreq.8.0 (Equation 5).

"[23]

"A virus-free protein drug obtained by a method according to any of [1] to [22].

"Advantageous Effects of Invention

"The present invention can provide a protein drug having a high rate of virus removal by a method for manufacturing a virus-free protein drug, comprising a step of filtering a virus-containing protein solution through a small-pore size virus removal membrane at a low filtration pressure. Thus, for example, in the case where virus-containing protein solutions are continuously filtered at a low filtration pressure, a post-wash step or a stop and start step are included, a protein drug having a high rate of virus removal can be provided.

BRIEF DESCRIPTION OF DRAWING

"FIG. 1 is a graph showing the relationship between the pH (X) and salt ionic strength (Y (mM)) of a solution prior to filtration in the absence of virus leakage in Example 2. Lines corresponding to the equations 1, 2, and 3 determined in Example 2 are indicated from left to right."

For more information, see this patent application: Hongo, Tomoko; Hayashida, Hirohisa. Method for Manufacturing Protein Drug. Filed June 22, 2012 and posted July 24, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2616&p=53&f=G&l=50&d=PG01&S1=20140717.PD.&OS=PD/20140717&RS=PD/20140717

Keywords for this news article include: Viruses, Peptides, Virology, Amino Acids, Blood Proteins, Biological Factors, Blood Coagulation Factors, Asahi Kasei Medical Co. Ltd..

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Source: Life Science Weekly


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