JP2890465B2 - Method for producing anti-NANB virus antibody-positive intravenous immunoglobulin preparation - Google Patents

Description

The present invention relates to a method for producing an anti-NANB (also referred to as non-A non-B hepatitis or hepatitis C) virus antibody-positive intravenous immunoglobulin preparation.

[Conventional technology]

As intravenous immunoglobulin preparations containing an antibody against a specific virus as a main component, those for various viruses (hepatitis B virus, cytomegalovirus, varicella-zoster virus, influenza virus, etc.) are known. Used for prophylactic treatment of viral infections.

[Problems to be solved by the invention]

The purpose of the present invention is to provide a clinically applicable formulation,
It is an object of the present invention to provide a method for producing an anti-NANB virus antibody-positive intravenous immunoglobulin preparation which is highly safe and effective.

[Means for solving the problem]

The present inventors have studied the industrial production method of anti-NANB virus antibody-positive intravenous immunoglobulin preparations for this purpose, and as a result, combined polyethylene glycol (hereinafter, referred to as PEG) fractionation treatment, heat treatment, etc. By setting processing conditions for each process, highly safe and effective
The present inventors have found that a NANB virus antibody-positive intravenous immunoglobulin preparation can be obtained, and have further studied to complete the present invention.

(1) Starting Material As a starting material of the present invention, a fraction containing an anti-NANB virus antibody-positive immunoglobulin is used, which is derived from human plasma and contains an anti-NANB virus antibody-positive immunoglobulin fraction. There is no particular limitation as long as it exists. Specifically, fraction II + III, fraction II, or anti-N obtained by ethanol fractionation of corn from anti-NANB virus antibody-positive plasma
Pastes of fractions equivalent to these containing ANB virus antibody-positive immunoglobulin and the like can be mentioned. The starting material may contain human blood group antibody, kallikrein, prekallikrein, IgM, IgG polymer, and the like. The confirmation of the positive is, for example, Glu-Phe-Arg-Glu-Gln-Asp-Gln-Ile-Lys-Thr
-Lys-Asp-Arg-Thr-Gln-Gln-Arg-Lys-Thr-Lys
-Arg-Ser-Thr-Asp-Arg-Arg-Arg-Ser-Lys-Asn
A peptide comprising the amino acid sequence of Glu-Lys-Lys-Lys-Lys-Lys-Glu-Phe is converted into a reagent by a known immunological method (RI method, RPHA method, EIA method, latex agglutination method); This is done by detecting the reactivity with. This positive fraction is used as a starting material. In addition, the EIA reagent was exemplified in Reference Examples described later.

(2) Production method The production method according to the present invention preferably comprises the following treatment.

Low concentration polyethylene glycol (PEG) treatment step This step is a step of treating the starting material with low concentration PEG and collecting the supernatant.

The starting materials are suspended in a suitable aqueous solvent. As a solute of the aqueous solvent, for example, sodium chloride, sodium phosphate, potassium phosphate, acetic acid, sodium acetate, citric acid, sodium citrate and the like may be contained.

This suspension is prepared with a molecular weight of 1,000 to 10,000 (preferably about 2,000
6,000 6,000) (eg, mixing both). As the processing conditions, the PEG concentration is 4 to 10 w / v% (particularly 4%).
-8w / v%), pH 4-6 (especially 4.5-5.5), ionic strength 0.0
It is preferably 001 to 0.1M (particularly 0.0001 to 0.01M).

At this time, the protein concentration is 1 to 20 w / v% (particularly, 5 to 15 w / v%)
It is preferred that

This treatment is carried out by stirring at about 0 to 4 ° C., usually for about 30 minutes to 6 hours.

Then, for example, centrifugation (6000-8000 rpm, 10-30
Minutes) and collect the supernatant.

High concentration PEG treatment step This step is a step of treating the supernatant obtained in the step with high concentration PEG and collecting the precipitate.

The above supernatant was used for a molecular weight of 1,000 to 10,000 (preferably 2,000 to 6,
000) (for example, both are mixed). The processing conditions include a PEG concentration of 10 to 15 w / v% (particularly about 11 to 13 w / v%), a pH of 6 to 9 (particularly 7.5 to 8.5), and an ionic strength of 0.0001 to 0.1 M (particularly 0.0001 to 0.01 M). Preferably, there is.

At this time, the protein concentration is 1 to 20 w / v% (particularly, 5 to 15 w / v%)
It is preferred that

This treatment is carried out by stirring at about 0 to 4 ° C., usually for about 30 minutes to 6 hours.

Then, for example, centrifugation (6000-8000 rpm, 10-30
Min) and collect the precipitate.

Anion exchanger treatment step This step is a step of dissolving the precipitate fraction obtained in the step in an aqueous solvent or contacting with an anion exchanger after the treatment in the step described below to collect a non-adsorbed fraction. is there.

This step is performed to remove IgM, IgA and IgG polymers.

(I) Preparation of anion exchanger The anion exchanger is obtained by bonding an anion exchange group to an insoluble carrier. Examples of the anion exchange group include a diethylaminoethyl (DEAE) group and a quaternary aminoethyl (QAE) group. And agarose, cellulose, dextran, polyacrylamide and the like can be used as the insoluble carrier.

 The coupling is performed by a known method.

(Ii) The precipitate fraction obtained in the step of the treatment method is dissolved in a suitable aqueous solvent. The aqueous solvent is pH 5-7 (preferably pH 5.5-6.5),
It is preferably an aqueous solution having a low ionic strength (preferably 0.0001 to 0.1 M). May be included. The protein concentration is 1 to 15 w / v% (particularly, 3 to 10 w / v
/ v%) is preferred.

Further, a contact treatment is performed with the anion exchanger equilibrated with the aqueous solvent. In the treatment, either a batch method or a column method may be used.

For example, in the batch method, 1 ml of the anion exchanger is mixed with about 10 to 100 ml of the solution to be treated,
After stirring for about 0 minutes to 2 hours, centrifuge (6,000 to 8,000 r
pm, 10-30 minutes) and collect the supernatant.

Also in the column method, about 10 to 100 ml of the solution to be treated is brought into contact with 1 ml of the anion exchanger to collect the non-adsorbed fraction.

Note that this step can be omitted if desired. In the case of performing liquid heat treatment, it is preferable to perform the anion exchanger treatment after the immobilized human blood group substance treatment.

Immobilized human blood group substance treatment step This step is a step of recovering the non-adsorbed fraction by contacting the precipitated fraction or the non-adsorbed fraction obtained in the step with the immobilized human blood group substance.

 This step is performed to remove human blood group antibodies.

(I) Preparation of immobilized human blood group substance The immobilized human blood group substance is immobilized on an insoluble carrier of human blood group substance.

A known method may be used for preparing a human blood group substance. For example, it can be obtained by subjecting human A, B, AB or O type erythrocytes to hemolysis or sonication in a hypotonic solution, followed by purification by ammonium sulfate fractionation or PEG fractionation.

Furthermore, after this human blood group substance is dissolved in physiological saline,
It is said to be effective for inactivating contaminating viruses, for example, at about 50 to 70 ° C., preferably about 60 ° C., for 7 to 13 hours, preferably for about 10 hours, or about 80 to 130 ° C., preferably 95 ° C.
Heat treatment at ~ 121 ° C for about 1-40 minutes, preferably about 2-30 minutes. Then, centrifugation is performed to remove insolubles, and dialyzed against distilled water to obtain each human blood group substance. Further, a synthetic antigen (sugar chain) can also be used as a human blood group substance. [See Chem. Soc. Rev. p423-452 (1978)] On the other hand, agarose, cellulose,
Dextran, silica gel, glass and the like are used.

Immobilization may be performed according to a known method. For example, agarose, cellulose and the like can immobilize a human blood group substance by the CNBr activation method, silica gel, glass and the like can be immobilized by the oxirane method.

(Ii) Treatment method The substance to be treated, for example, a solution obtained by dissolving a precipitate fraction from the step (1) in an aqueous solvent or a non-adsorbed fraction from the step (3)
-8 (especially pH 6-7), ion concentration 0.0001-0.1M (especially
(0.0001 to 0.01 M) under the condition of contact with the immobilized human blood group substance equilibrated with the above aqueous solvent. At that time, the protein concentration is preferably 1 to 15 w / v% (particularly, 3 to 10 w / v%), and either the batch method or the column method may be used.

For example, in the batch method, 1 ml of the immobilized human blood group substance is mixed with about 10 to 100 ml of the solution to be treated, and 0 to 10
° C, preferably at 0-4 ° C for 30 minutes-4 hours, preferably
After stirring for about 30 minutes to 2 hours, centrifuge (6,000 to 8,000
rpm, 10-30 minutes) and collect the supernatant.

Also in the column method, about 10 to 100 ml of the solution to be treated is brought into contact with 1 ml of the immobilized human blood group substance, and the non-adsorbed fraction is collected.

 This step can be omitted if desired.

Heat treatment step This step minimizes the decrease in the antibody activity of the immunoglobulin against the NANB virus in the presence of the stabilizer at the desired stage, but completely eliminates contaminating viruses such as HB virus and NANB virus. This is a step of performing a heat treatment under inactivating conditions. The heat treatment is performed in a dry state having a humidity of 3% or less (that is, a dry treatment) or in a solution state, that is, in an aqueous state of an immunoglobulin (that is, a liquid heat treatment).
More preferably, liquid heat treatment is recommended.

As a stabilizer, a disaccharide (eg, saccharose, maltose) and a sugar alcohol (eg, sorbitol, mannitol) are preferably exemplified in any treatment. More preferred is sorbitol.

In the dry heat treatment method, the amount of the stabilizer added is 0.5 to 5 w / v% (preferably, 1 to 3 w / v%) of disaccharides, sugar alcohols, and the like.
In liquid heat treatment, disaccharides, sugar alcohols, etc. are 10w / v%
Use of the above (preferably 20 to 45 w / v% or 10 to 35 w / w%) is suitably exemplified.

The amount of immunoglobulin to be heated is 1 to 10 w / v% (preferably 3 to 7 w / v%) as a protein in the dry heat treatment.
%) Is preferably adjusted. In the liquid heat treatment, the protein amount is 0.1 to 30 w / v% (preferably 5 to 30 w / v%).
It is preferably adjusted to 20 w / v%).

In the case of dry heat treatment, after adding a stabilizer,
If necessary, sterilization filtration is performed, and the moisture content is reduced to 3% or less, preferably 1% or less, for example, by freeze-drying. Examples of the freeze-drying conditions include a vacuum of 0.5 mmHg at 20 to 40 ° C. for about 24 to 96 hours. Then, for example, the treatment is performed at 50 to 70 ° C. (preferably about 60 ° C.) for 10 to 200 hours (preferably about 50 to 100 hours).

Further, by performing this heat treatment step in an inert gas atmosphere, stability during heating can be further improved.
Examples of the inert gas include nitrogen gas, argon, and helium.

In the case of liquid heat treatment, the pH of the aqueous solution is adjusted to 4.5 to 6.5, preferably to pH 5 to 6, and in the liquid heat treatment method, for example, 50 to 50 is used.
The treatment is carried out at a temperature of about 70 ° C. (preferably about 60 ° C.) for 10 minutes to 20 hours (preferably about 10 hours).

In the case of dry heat treatment, the heat treatment step is preferably performed in the final step.

In the case of the liquid heat treatment, it is preferable to perform the heat treatment on the starting material or after the step.

In addition, when it performs after the process of, it is more preferable to perform the process of and again from the point of a foreign substance removal.

Further, if necessary, other known means (for example, treatment by protection of immobilized diamino) may be performed.

After completion of all the steps, a liquid preparation can be prepared by a known method, that is, dialysis, sterilization filtration, dispensing, or the like. Furthermore, a dry preparation can be obtained by an operation such as freeze-drying.

In the case of a liquid preparation, the globulin concentration is 1 to 15 w / v
% (Preferably 5 to 10 w / v%). It is preferable to add a stabilizer. Examples of the stabilizer include sugars and sugar alcohols. Specific examples thereof are the same as those already disclosed in the heat treatment.

The amount of the stabilizer added is globulin 1 to 15 w / v%
Per unit, about 1 to 10 w / v%, preferably about 5 w / v%.

The pH of the liquid preparation is about 5.3 to 5.7, preferably 5.5.
For example, it is set as the degree.

In the case of a dry preparation, it is preferable to add a stabilizer. Examples of the stabilizer include sugar, sugar alcohol, albumin, inorganic salt and the like. Specific examples thereof are the same as those already disclosed in the heat treatment for sugar and sugar alcohol, and sodium chloride is exemplified as an inorganic salt.

The amount of the stabilizer added is about 1 to 10 parts by weight (preferably 2 parts by weight) of sugar or sugar alcohol and 0.5 to 5 parts by weight (preferably 1 part by weight) of albumin per 1 to 15 parts by weight of globulin. And about 0.1 to 1 part by weight (preferably 0.5 part by weight) of inorganic salt.

[Action / Effect]

The preparation obtained according to the present invention has almost no immunoglobulin inactivated, and further, does not contain impurities such as anti-human blood group substance antibodies and is subjected to heat treatment, so that contaminating viruses are inactivated, and It is a safe preparation that has properties such as sufficiently low complement activity and can meet the Japanese Standards for Biological Preparations (hereinafter referred to as “raw standards”). Non-chemically modified immunoglobulin preparations are preferred.

 In the case of a dry preparation, the solubility is good.

When the preparation obtained by the present invention is used, when it is a liquid preparation, it can be used as it is, or diluted with an appropriate solvent (for example, distilled water for injection, physiological saline, glucose solution, etc.),
For dry preparations, a suitable solvent (eg, distilled water for injection)
And used for prevention or treatment of NANB virus-derived hepatitis by intravenous administration, infusion, or the like. It can also be used in combination with other known hepatitis prophylactic / therapeutic agents, especially NANB prophylactic / therapeutic agents.

[Examples, Reference Examples, Experimental Examples]

Examples and experimental examples are described in order to explain the present invention in more detail, but the present invention is not limited to these examples.

Example 1 Anti-N screened using the EIA reagent obtained in Reference Example
ANB virus antibody-positive plasma was added to 1 kg of the corn fraction II + III obtained by the cold ethanol method, and cold water 10 was added thereto to adjust the pH to 5.5.
Then, PEG # 4000 was added to a final concentration of 8%, and centrifuged at 2 ° C.

The resulting supernatant was adjusted to pH 8.0 using 1N-sodium hydroxide.
After that, PEG # 4000 was added to a final concentration of 12%, and centrifugation was performed at 2 ° C. to collect an IgG fraction.

This IgG fraction was dissolved using a 0.6% sodium chloride solution so that the IgG concentration became 7%, and the pH was adjusted to 6.5.

100 ml of this IgG solution was passed through 3 ml of a separately prepared human blood group substance formyl cellulofine column to adsorb and remove human blood group antibodies. Blood group antibody was reduced from (1:32) to (1: 2) by adsorption in this step.

To this unadsorbed fraction, human albumin (1 w / v%) and saccharose (2 w / v%) were added per IgG5 w / v% solution, sterilized, filtered and lyophilized.

After freeze-drying, heat treatment was performed at 60 ° C for 72 hours to obtain an intravenous immunoglobulin preparation.

This formulation comprises substantially only IgG monomeric, human blood group antibodies amount sufficient small, anti-complement activity was also about 10~15CH _50.

In addition, the solubility was high, and it passed the raw standard as an immunoglobulin for intravenous injection.

In addition, this immunoglobulin suppressed the growth of NANB virus in a test using chimpanzees.

Example 2 Anti-N screened using the EIA reagent obtained in Reference Example
Water 10 was added to 1 kg of the corn fraction II + III obtained from the ANB virus antibody-positive plasma by the cold ethanol method, and
After adding 50 g of sorbitol per 00 ml and adjusting the pH to 5.5, a heat treatment was performed at 60 ° C. for 10 hours.

After the heat treatment, the pH was adjusted to 5.5, PEG # 4000 was added to a final concentration of 6%, extraction was performed at 2 ° C. for 3 hours, and then centrifugation was performed at 2 ° C.

The resulting supernatant was adjusted to pH 8.8 using 1N-sodium hydroxide, and then PEG # 4000 was added to a final concentration of 12%.
Centrifugation was performed at 2 ° C., and an IgG fraction was obtained as a precipitate fraction. The precipitate was dissolved in distilled water, and 100 ml of this IgG solution was passed through a 3 ml human blood group substance formyl cellulofine column equilibrated with distilled water to adsorb and remove human blood group antibodies. Due to the adsorption in this step, the blood group antibody is changed from (1:32) to (1:32).
2) decreased. The precipitate was equilibrated with 0.4% saline
Add AE-Sephadex (2 ml per 50 ml solution) and add 0
Contact treatment at ~ 4 ° C for about 1 hour, and filtration after treatment
The filtrate (IgG solution) was recovered except for DEAE-Sephadex.

After adding and dissolving 2% sorbitol, 0.5% NaCl and 1% albumin to the IgG fraction to adjust the pH to 6.8, the mixture was sterilized and filtered.
It was freeze-dried to obtain an intravenous immunoglobulin preparation.

This formulation comprises substantially only IgG monomeric, human blood group antibodies amount sufficient small, anti-complement activity was also about 10~15CH _50.

In addition, the solubility was high, and it passed the raw standard as an immunoglobulin for intravenous injection.

Example 3 In Example 2, after treatment with DEAE-Sephadex, the mixture was centrifuged (7000 rpm, about 20 minutes) and the supernatant (IgG solution) was obtained.
Was recovered.

This IgG solution was adjusted to a 5% IgG solution with distilled water, the pH of the solution was adjusted to about 5.5 with sodium acetate, and sorbitol was further added to a final concentration of 5%. This aqueous solution (conductivity about 1mmh
o) was sterilized and filtered to obtain a liquid immunoglobulin preparation for intravenous injection.

This formulation comprises substantially only IgG monomeric, human blood group antibodies amount sufficient small, anti-complement activity is also about 10～15CH _50, was also passed to the raw reference as intravenous immunoglobulin .

In a test using chimpanzees, the growth of NANB virus was suppressed.

Example 4 Anti-N screened using the EIA reagent obtained in Reference Example
After heating the ANB virus antibody-positive plasma at 56 ° C. for 30 minutes, the corn fraction II was collected by the cold ethanol method as described below.

The supernatant was collected after treatment with ethanol concentration of 8%, pH 7.2, -3 ° C for 16 hours, and the precipitate (corn fraction II + III) was treated by treatment with ethanol concentration of 21%, pH 6.8, -5 ° C for 24 hours. The precipitate was dissolved and the ethanol concentration was 20%, pH 6.6, -5
The precipitate was dissolved by treating at 16 ° C for 16 hours, and the precipitate was dissolved.
Collect at 8 ° C for 8 hours, and collect the supernatant (ethanol concentration 25%, pH 7.4, -5 ° C, 16 hours) to collect the precipitate (corn fraction II). After suspending at 10 and adjusting the pH to 5.5, centrifugation was performed, and the supernatant was recovered.
Heat treatment was performed at 10 ° C. for 10 hours.

After the heat treatment, the pH was adjusted to 5.5, PEG # 4000 was added to a final concentration of 6%, and the mixture was centrifuged at 2 ° C.

The obtained supernatant was adjusted to pH 8.0 using 1N-sodium hydroxide, and PEG # 4000 was added to a final concentration of 12%.
Centrifugation was performed at 2 ° C., and an IgG fraction was obtained as a precipitate fraction.

Reference Example EIA reagent (1) Preparation of immobilized peptide A microplate was prepared, and the synthetic peptide Glu-Phe-Arg-Glu-Gln-Asp-Gln-Ile-Lys-Thr was placed in each well of the microplate.
-Lys-Asp-Arg-Thr-Gln-Gln-Arg-Lys-Thr-Lys
-Arg-Ser-Thr-Asp-Arg-Arg-Arg-Ser-Lys-Asn
-Glu-Lys-Lys-Lys-Lys-Lys-Glu-Phe 0.05 M sodium bicarbonate solution was poured in 100 μl each,
It was left at 4 ° C. for 1 hour. Then, the plate was washed with a 0.9% sodium chloride solution containing 0.05% Tween-20, and 200 μl of an isotonic phosphate buffer (pH 7.2) containing 0.25% bovine serum albumin was poured into each well. Thereafter, the mixture was left at room temperature for 3 hours to remove the solvent, thereby preparing an immobilized NANB-related peptide.

(2) Preparation example of enzyme-labeled antibody A peroxidase was prepared by binding to an anti-human IgG antibody by a usual glutaraldehyde method.

(3) Measurement Example First, the microplate prepared in (1) was prepared, and 75 μ of an isotonic phosphate buffer containing 1% rabbit serum and 0.25% bovine serum albumin was poured into each well. Further, 25 μl of the sample solution was added to each well, and left at room temperature for 1 hour. Then, 0.05% Tween-20 was added.
A diluted solution obtained by washing with a 9% sodium chloride solution and diluting the enzyme-labeled antibody prepared in (2) with an isotonic phosphate buffer (pH 7.2) containing 1% rabbit serum and 0.25% bovine serum albumin. Was added to each well at 100 μl, left at room temperature for 1 hour, and washed with a 0.9% sodium chloride solution containing 0.05% Tween-20. And contained 1 mg / ml of orthophenylenediamine and 0.015% hydrogen peroxide.
Add 0.05 M citrate-phosphate buffer (pH 5) to each well.
0μ was added and left at room temperature for 30 minutes. Further, 100 μl of 2N sulfuric acid was added to each well, and this was subjected to absorbance measurement.

The absorbance was measured at a wavelength of 492 nm, and these absorbances were measured. The control absorbance was defined as the absorbance at 690 nm, and the enzyme activity was determined by calculating the relative ratio of these absorbances (A ₄₉₂ / A ₆₉₀ ). The same procedure was applied to the negative serum to determine the enzyme activity (Blank value). A sample showing a value twice or more of the blank value was determined to be positive.

In addition, a standard calibration curve is prepared by a reaction system using a standard product, and by comparing the enzyme activity obtained in the reaction of the test solution with the calibration curve, the amount of the anti-NANB virus antibody contained in the test solution is determined. I asked.

Experimental Example 1: Measurement of antibody titer Preparation method Antibody-positive plasma was asymptomatic and obtained in Reference Example
Negative plasma was collected from normal individuals from NANB virus antibody positive carriers screened using EIA reagents. Intravenous immunoglobulins were obtained from these plasmas by the method of Example 4. The IgG content of this fraction was determined by the immunodiffusion method (MBL Co., Nagoya), and the antibody titer was determined by the method of the above reference example (Table 1).

Continuation of the front page (56) References JP-A-63-183539 (JP, A) JP-A-56-73029 (JP, A) JP-A-2-186990 (JP, A) Kuo, et al. , Science, 244, Apr. 1989, pp. 284-143. 362- 364 (58) Fields investigated (Int. Cl. ⁶ , DB name) A61K 39/395 A61K 39/29 CA (STN) REGISTRY (STN) WPIDS (STN) MEDLINE (STN)

Claims (5)

(57) [Claims] (1) Formula (I): Starting from a human plasma-derived fraction containing an anti-NANB virus antibody-positive immunoglobulin having reactivity with a peptide consisting of the amino acid sequence represented by
Method for producing NANB virus antibody-positive intravenous immunoglobulin preparation: (a) pH 4 to 6, ionic strength 0.0001 to 0.1M, temperature 0 to 0
The supernatant is recovered by treating with 4 to 10 w / v% polyethylene glycol having a molecular weight of 1,000 to 10,000 under the condition of 4 ° C. (B) The supernatant of (a) was used at pH 6-9, ionic strength 0.0001-
The precipitate is recovered by treatment with polyethylene glycol having a molecular weight of 1,000 to 10,000 at 10 to 15 w / v% under conditions of 0.1 M and a temperature of 0 to 4 ° C. (C) heat-treating in the presence of a stabilizer under conditions sufficient to inactivate contaminating viruses in the desired step. 2. The method according to claim 2, further comprising, after the step (b), a step of treating with an immobilized human blood group substance under conditions of pH 5 to 8 and ionic strength of 0.0001 to 0.1 M to collect a non-adsorbed fraction. The manufacturing method according to claim 1, wherein 3. The precipitate of (b) is dissolved in an aqueous solvent,
7. The production method according to claim 1, further comprising a step of recovering a non-adsorbed fraction by treating with an anion exchanger under conditions of an ionic strength of 0.0001 to 0.1M. 4. Formula (I): Manufactured using a human plasma-derived fraction containing an anti-NANB virus antibody-positive immunoglobulin having reactivity with a peptide consisting of an amino acid sequence represented by the starting material, substantially Ig
An anti-NANB virus antibody-positive intravenous immunoglobulin preparation containing only G monomer and substantially no infectious virus. 5. Formula (I): EIA using a peptide consisting of the amino acid sequence represented by
The immunoglobulin preparation according to claim 4, which has an antibody titer of at least 51200 when measured with a reagent.