TWI585101B - Method for preparing antibody f(ab')2 fragments - Google Patents

Description

Method for preparing antibody F(ab') 2 fragment

The present invention relates to a method for preparing an antibody F(ab') ₂ fragment, in particular to a method using a cation exchange resin for purification without precipitation with ammonium sulfate or sodium sulfate to obtain a high recovery, high purity antibody F (ab ') Preparation method of ₂ fragments.

Since antibodies can specifically bind to the characteristics of antigens, many antibodies have been used for therapeutic purposes, such as treatment against infectious diseases (eg, infections such as bacteria, viruses, parasites, etc.), cancer, drugs, or other foreign bodies. In the structure of the antibody to IgG as an example, antibodies can be divided into F (ab ') ₂ and Fc portions of two, F (ab') via hydrolysis with pepsin (Pepsin) of ₂ disulfide bonds to two antigen The binding site has a molecular weight of approximately 110 kDa.

Since the F(ab') ₂ fragment removes the Fc portion, it is possible to eliminate the non-existence between the antibody and the Fc receptor of cells such as macrophages, dendritic cells, neutrophils, NK cells, and B cells. It has a specific sensitivity and is more effective in infiltrating the tissue due to its smaller molecule. In addition, since the Fc portion is removed, it can be detected without interference from the anti-Fc antibody, and the application has higher sensitivity in detection. Better than IgG in terms of safety and efficacy.

The production of antibody F(ab') ₂ fragments for therapeutic use often involves many steps in maintaining their effectiveness while at the same time reducing the incidence and severity of side effects by purification. These steps include ammonium sulfate or sodium sulfate precipitation, enzymatic digestion, thermocoagulation, diafiltration, and more recently ion exchange and affinity column chromatography. Among them, a more delicate method is to purify the antibody from the serum, continue to be hydrolyzed by protease to produce the F(ab') ₂ fragment, and then the F(ab') ₂ fragment and the Fc hydrolyzate by precipitation and column chromatography. Protease separation. There are also self-serums which are first hydrolyzed by proteases and then precipitated by fractional ammonium sulfate or sodium sulfate plus chromatography column analysis. These preparative steps result in an effective and safe F(ab') ₂ product, but the cost of preparation is quite high. In addition, the step of precipitating ammonium sulfate or sodium sulfate in the process will be quite time consuming and the recovery rate is relatively high, resulting in a substantial increase in manufacturing costs. In order to avoid the ammonium sulfate precipitation step, the related research has applied Q-Sepharose column plus Tangential flow diafiltration to purify F(ab') ₂ without the use of ammonium sulfate precipitation (Jones et al . Journal of Immunological Methods (2003) 275 (1-2), 239-250). However, when the purification method is applied, the state of hydrolysis needs to be monitored, so that the hydrolysis product of the antiserum except F(ab') ₂ and other components are less than 13 kDa for purification purposes. In addition to being prone to excessive hydrolysis, this condition may only be applied to specific serum samples. The sample used in the report, IgG is the main component of serum protein, and the content is greater than the sum of other protein (albumin-containing) components.

Therefore, there is still a need to continuously improve the method of preparing the antibody F(ab') ₂ fragment to shorten the preparation time, reduce the preparation cost, and improve the purification recovery rate to obtain a high purity and quality antibody F(ab') ₂ fragment.

The present invention provides a method of preparing and purifying an antibody F(ab') ₂ fragment, and a pharmaceutical composition comprising the antibody F(ab') ₂ fragment prepared using the method.

In one aspect, the invention provides a method of preparing an antibody F(ab') ₂ fragment, comprising the steps of: (1) hydrolyzing an antibody with pepsin to obtain an antibody F(ab') ₂ fragment; The antibody F(ab') ₂ fragment is concentrated in a filtration system and replaced with a buffer wherein the replacement of the buffer causes the antibody F(ab') ₂ fragment to have a positive charge; and (3) the antibody F(ab') _{The 2} fragment was purified by a cation exchange column and the purified antibody F(ab') ₂ fragment was replaced with a saline solution. The purified antibody F(ab') ₂ fragment does not contain serum albumin, intact antibody molecules and antibody Fc fragments.

Preferably, the purified antibody F(ab') ₂ fragment can be concentrated by a second filtration system and the second buffer replaced with the requirements of use.

In one embodiment of the invention, the filtration system can be a Tangential Flow Filtration System.

In one embodiment of the invention, the pH of the buffer is less than the isoelectric point (pI) value of the F(ab') ₂ fragment of the antibody, for example, the buffer may be a sodium acetate solution at pH 4.3.

In one embodiment of the invention, the aqueous salt solution can be a sodium chloride (NaCl) aqueous solution having a concentration of from about 0.1 M to about 2 M.

In a specific embodiment of the invention, the second buffer may be physiological saline.

In one embodiment of the invention, the second filtration system can be a Tangential Flow Filtration System.

In a particular embodiment of the invention, the antibody can be an immunoglobulin, for example, IgG, IgA or ÍgM.

In another aspect, the present invention provides a composition comprising an antibody F(ab') ₂ fragment, wherein the antibody F(ab') ₂ fragment is produced by the method of the present invention, and the composition does not contain serum white Protein, intact antibody molecule and antibody Fc fragment.

In still another aspect, the present invention also provides a pharmaceutical composition comprising an antibody F(ab') ₂ fragment, accompanied by one or more pharmaceutically acceptable carriers, wherein the antibody F(ab') ₂ fragment is described in the present invention The method is prepared, and the pharmaceutical composition does not contain serum albumin, intact antibody molecules, and antibody Fc fragments.

These and other aspects of the present invention will be apparent from the following description of the preferred embodiments of the invention and Modification.

All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, unless otherwise defined. In case of conflict, this document, including its definition, is the main one.

As used herein, the articles "a" or "the" are used to mean that the grammatically accepted words are one or more (ie, at least one). For example, "an element" means one element or more than one element.

The terms "about", "about", or "approximately" as used herein shall generally mean within 20% of the specified value or range, preferably within 10%, and more preferably within 5%. The quantities given herein are approximate, meaning that the terms "about", "about", or "approximately" may also be inferred if not explicitly stated.

An antibody, also known as an immunoglobulin, which is present in the serum of an individual and is part of the immune system, by binding to a specific antigen and neutralizing it, and inducing a subsequent immune response to protect the individual from foreign substances, For example, pathogens, invasive functions. Due to the specific binding properties between antigen and antibody, there are various applications in the industry, such as testing, detecting, preventing and treating diseases. Among the currently known antibodies, for example, IgG, ÍgM, IgA, IgE, IgG is the highest in blood, and IgG is widely used as a product of a mature immune reaction.

When IgG is decomposed by an enzyme, different products can be obtained depending on the enzyme used. For example, when using papain, IgG is decomposed into an Fc fragment (crystallizing fragment) and two Fab fragments (antigen-binding fragment). If pepsin is used, an F(ab') ₂ fragment and an Fc fragment can be obtained. The Fab fragment and the F(ab') ₂ fragment still retain the property of specifically binding to the antigen, while the F(ab') ₂ fragment has a function of precipitating the antigen. In contrast, the Fc fragment plays the role of a signal marker, attracting and activating macrophages, neutrophils, etc. to recognize and phagocytose antigen-antibody complexes. In addition, when a heterologous antibody enters an individual, the Fc fragment is regarded as a foreign substance due to a specific sequence of its epitope, thereby inducing a serious immune reaction and causing side effects. Therefore, the antibody is currently treated with an enzyme in industrial utilization. The Fc fragment, the Fab fragment and the F(ab') ₂ fragment are removed, and since the molecules of the Fab fragment and the F(ab') ₂ fragment are smaller, it is more effective to infiltrate into the tissue to improve the efficacy.

At present, most of the preparation methods for F(ab') ₂ fragments in the industry are to first purify the antibody and then excise the Fc by protease hydrolysis, and then separate the product by affinity column or other characteristic chromatography. Such methods operate at a lower risk, but the process is longer and costly and is generally only used in small batches in research laboratories. When applied to a large-scale preparation process, the p(ab') ₂ fragment and other hydrolysates are mostly separated by pepsin, and the F(ab') ₂ fragment is coarsely separated by ammonium sulfate precipitation, but this step is a time-consuming task. Generally, precipitation, solid-liquid separation and re-dissolution dialysis of sediments take at least 2 working days, and the equipment and filter consumables required for large-scale operation in solid-liquid separation are also very expensive. Moreover, the purity and recovery of the F(ab') ₂ fragment in ammonium sulfate precipitation is a dilemma. The 13% ammonium sulfate commonly used in the industry can obtain a relatively pure F(ab') ₂ fragment but the recovery rate is less than 50%. 45% ammonium sulfate precipitates the F(ab') ₂ fragment almost but cannot be separated from the rest of the protein. In addition, the crude separation with ammonium sulfate precipitation still requires further purification by anion exchange resin.

In order to overcome the dilemma of ammonium sulfate precipitation, Jones and Landon have reported the hydrolysis of protein in serum or plasma to small peptides, and then dialysis to isolate F(ab') ₂ fragments (Jones et al., Journal of Immunological Methods) (2003) 275 (1-2), 239-250). However, in this method, it is necessary to continuously monitor the hydrolysis reaction of pepsin, and have two reaction endpoints to be observed, one of which is that the antibody needs to be hydrolyzed to F(ab') ₂ , but excessive hydrolysis causes F(ab') ₂ The yield is reduced or even inactivated; the other is to control the hydrolysis of other proteins in serum or plasma into small peptides (13 kDa), which are difficult to achieve at the same time, so there are still limitations in industrial use. .

In view of the above, the present invention provides a method for preparing an antibody F(ab') ₂ fragment, which comprises the steps of: (1) hydrolyzing an antibody with pepsin to obtain an antibody F(ab') ₂ fragment; The antibody F(ab') ₂ fragment is concentrated in a filtration system and replaced with a buffer wherein the replacement of the buffer causes the antibody F(ab') ₂ fragment to have a positive charge; and (3) the antibody F(ab') _{The 2} fragment was purified by a cation exchange column and the purified antibody F(ab') ₂ fragment was replaced with a saline solution. The purified antibody F(ab') ₂ fragment does not contain serum albumin, intact antibody molecules and antibody Fc fragments. Preferably, the purified antibody F(ab') ₂ fragment can be concentrated by a second filtration system and replaced with a second buffer as needed. Thus, the above method can not only overcome the low recovery rate or low purity caused by the ammonium sulfate precipitation step, but also shorten the preparation time, reduce the preparation cost, and improve the purification recovery rate, thereby obtaining the high purity and quality antibody F(ab'. ) ₂ fragments.

The term "antibody" as used herein refers to an intact antibody, and "antibody fragment" refers to an antigen-binding fragment comprising an F(ab') ₂ fragment in an intact antibody, which still retains the function of specifically binding to an antigen and can replace the intact antibody. Features. In one embodiment, the antibody and antibody fragments can be made by recombinant DNA techniques. In another embodiment, the antibody and antibody fragments can be obtained from natural sources, wherein the antibody fragments can also be obtained by chemical or enzymatic action of intact antibodies.

The pepsin effective amount obtained by hydrolyzing the antibody to obtain the F(ab') ₂ fragment can be determined by the amount of the antibody which is hydrolyzed by a person having ordinary knowledge in the art, and the activity of pepsin. In a specific embodiment of the present invention, The final concentration of pepsin was 0.05% (w/v).

In a particular embodiment of the invention, the buffer having a positive charge on the F(ab') ₂ fragment of the antibody is acidic, for example, having a pH of between about 2.0 and 6.0, preferably between 3.0 and 5.0. Anyone skilled in the art will appreciate that any buffer system that can positively charge an F(ab') ₂ fragment of an antibody can be used, including but not limited to acetic acid, citric acid, phosphoric acid, 2- 2-morpholinoethanesulfonic acid (MES).

In another aspect, the present invention provides a composition comprising an antibody F(ab') ₂ fragment, wherein the antibody F(ab') ₂ fragment is produced by the method of the present invention, and the composition does not contain serum white Protein, intact antibody molecule and antibody Fc fragment.

Wherein the composition may further comprise a carrier for prolonging the half-life of the F(ab') ₂ fragment of the antibody, such as but not limited to a linear polymer (eg, PEG), a branched polymer, a lipid, cholesterol, a saccharide, Oligosaccharides, as well as synthetic or naturally occurring proteins, peptides, peptides.

In still another aspect, the present invention also provides a pharmaceutical composition comprising an antibody F(ab') ₂ fragment, accompanied by one or more pharmaceutically acceptable carriers, wherein the antibody F(ab') ₂ fragment is described in the present invention The method is prepared, and the pharmaceutical composition does not contain serum albumin, intact antibody molecules, and antibody Fc fragments.

For use in prophylaxis and therapy, the F(ab') ₂ fragment of the invention or a derivative thereof can be formulated into a pharmaceutical composition with a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" as used herein means that the carrier is compatible with the active ingredient contained in the composition, preferably to stabilize the active ingredient without causing harm to the subject to which the pharmaceutical composition is administered. The carrier can be a diluent, carrier, excipient or vehicle for the active ingredient. Examples of suitable carriers include physiologically compatible buffers such as Hank's solution, Ringer's solution, physiological saline buffer, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate , seaweed gum, tragacanth, gelatin, calcium citrate, microcrystalline cellulose, polyvinyl bromo ketone, cellulose, sterile water, syrup and methyl cellulose. The pharmaceutical composition may additionally comprise a lubricant, for example, talc, magnesium stearate and mineral oil; a wetting agent; an emulsifying and suspending agent; a preservative, for example, methyl- and propyl-hydroxybenzoate; sweet Flavor; and flavoring agent.

The pharmaceutical composition according to the present invention may be in the form of tablets, pills, powders, tablets, pouches, sachets, medicinal liquors, suspensions, emulsions, solutions, syrups, soft gelatin capsules and hard gelatin capsules, suppositories, sterile injectable solutions. And the form of the packaged powder.

The pharmaceutical compositions of the invention can be delivered via any physiologically acceptable route. Such routes include, but are not limited to, non-oral administration, systemic administration, oral administration Medicine, nasal administration, rectal administration, intraperitoneal injection, vascular injection, subcutaneous injection, transdermal administration, inhalation administration, and intramuscular injection.

The invention is further illustrated by the following examples, which are provided for purposes of illustration and not limitation. It will be apparent to those skilled in the art that <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt;

Example Materials and methods Antibody preparation

Pigs were immunized with Canine distemper virus (CDV) and boosted by the booster immunization of the pigs. Pig blood samples were collected. If the plasma was required, the blood sample was slowly injected into a blood collection bag containing 3.2% sodium citrate solution to a final volume ratio of 9:1, and the blood was mixed with the anticoagulant by shaking for several minutes. If the demand is serum, the blood sample is injected into a sterile test tube, placed obliquely for 10-30 minutes, and the blood is coagulated and collected to avoid hemolysis affecting subsequent analysis. The above sample was centrifuged to separate the supernatant and the solid, and the supernatant was obtained as a plasma sample or a serum sample, respectively.

F(ab’) ₂ Fragment production Pepsin treatment

The serum or plasma containing the anti-CDV antibody obtained through the above procedure was heated at 58 ° C for 30 minutes to deactivate the virus, followed by pepsin hydrolysis or cryopreservation at -80 ° C until use.

Before performing the pepsin hydrolysis reaction, 1 L of serum or plasma containing anti-CDV antibody is added to an equal volume of ultrapure water for dilution, followed by 10% pepsin (SIGMA-ALDRICH) previously dissolved in 0.1 N HCl to the final concentration. 0.05% (w/v), then slowly infused with 5N HCl into the above solution to adjust The pH is adjusted to about pH 3.2 ± 0.1. The pepsin-containing pig serum (pulp) solution was subjected to a hydrolysis reaction at 37 ° C for 2 hours, and then the solution was placed in a refrigerator at 4 ° C to terminate the reaction for subsequent dialysis.

Concentration and buffer replacement

The pepsin-hydrolyzed porcine serum (plasma) solution is then concentrated and buffer-replaced using a Tangential Flow Filtration System, where the tangential flow filtration system uses a pore size of 50 kD and a membrane area of 0.11 m. Membrane of ² (GE Healthcare Life Sciences; Kvick Lab Cassette-UFELA0050010ST). The porcine serum (pulp) solution was concentrated to 50% by volume, and then dialyzed against 10 volumes of 50 mM sodium acetate solution (pH 4.3), and the serum (plasma) solution sample after dialysis was continuously monitored until the conductivity was 50. Stop after mM sodium acetate solution is similar (less than 5 ms/cm). The collected solution was then sterile filtered through a 0.22 μm filter.

Purification of antibody F(ab') by cation exchange column ₂ Fragment

The cation exchange column SP-Sepharose FF (50 x 125 mm) was first equilibrated with 50 mM sodium acetate solution (pH 4.3), and the aforementioned pepsin was hydrolyzed and dialyzed to concentrate the porcine serum (plasma) solution with the replacement buffer. After entering the column, the column was washed with 50 mM sodium acetate solution (pH 4.3) to wash out the unbound, and the absorbance of the eluate at 280 nm was continuously monitored, and the absorbance near the column balance was judged as termination. Then, the desired F(ab') ₂ fraction was washed out with a 50 mM sodium acetate solution (pH 4.3) containing 0.2 M NaCl, and the heteroprotein was washed out with a 50 mM sodium acetate solution (pH 4.3) containing 1 M NaCl. The column was finally re-used with 0.1 M NaOH for in situ cleaning (CIP).

Dialysis replacement F(ab') ₂ Dosage buffer

The cation exchange column 0.2 M NaCl elution solution was collected, followed by a tangential flow filtration system (Tangential Flow Filtration Systems) for concentration and displacement buffer. The tangential flow filtration system used here a pore size of 50 kD, and the membrane area was 0.11 m ² of membrane enthalpy (GE Healthcare Life Sciences; Kvick Lab Cassette-UFELA 0050010ST). After the 0.2 M NaCl eluate was concentrated to 50% by volume, it was dialyzed against 10 volumes of physiological saline for injection. The collected solution was then sterile filtered through a 0.22 μm filter.

Antibody F(ab') ₂ Fragment virus neutralizing antibody titer test

To determine the antibody valence of the antibody F(ab') ₂ fragment, 50 μl of CDV virus (about 200 TCID ₅₀ ) was mixed with 50 μl of different dilutions for 1 hour, and the mixture was added to the preculture. B95-8 cell line was observed for cytopathic effect (CPE), and the highest dilution factor that did not produce cytopathic effects was used as the neutralizing antibody titer of the sample. When the sample is serum or plasma, it needs to be placed at 56 ° C for 30 minutes to deactivate the complement before testing.

result Example 1: Obtaining an extremely high purity antibody F(ab') via a cation exchange resin ₂ Fragment

The porcine anti-CDV antibody is hydrolyzed by pepsin to obtain the F(ab') ₂ fragment, and after dialysis by a tangential flow filtration system to concentrate and displace the buffer, the positively charged F(ab') fragment is further cationized. The exchange resin is separated and purified. The chromatogram of the separation and purification is shown in Fig. 1. The peak marked as unbound represents the substance not combined with the column, and then the column is washed with 0.2 M and 1.0 NaCl respectively to bond the tube. The column material was washed out and finally the column was washed with 0.1 M NaOH.

The eluate of each of the above stages was collected and analyzed by SDS-PAGE. The results of SDS-PAGE staining with Coomassie blue are shown in Fig. 2. The first lane shows the unhydrolyzed and purified plasma containing porcine anti-CDV antibody, wherein PSA represents porcine serum albumin, and the molecular weight of IgG antibody is about 135 kDa; the second channel is the product of the above-mentioned plasma pepsin hydrolysis, the molecular weight of each protein is significantly reduced; the third channel is the result of the above hydrolysis product through 50 kDa ultrafiltration membrane dialysis displacement buffer, compared with the use Before the ultrafiltration membrane was dialyzed, the protein composition distribution did not differ from that of the second lane, indicating that the ultrafiltration membrane dialysis could not obtain the separation and purification effect; the fourth lane was the cation exchange column filter; the last was the cation exchange column. After washing the column eluted with 0.2 M NaCl, the purified antibody F(ab') ₂ fragment was observed, and the degree of protein purification was significantly increased.

The purified material was then confirmed by Western blot analysis, and the rabbit anti-porcine IgG F(ab') ₂ fragment antibody (purchased from NOVUS) was used here, and the results are shown in Fig. 3, wherein the first lane contains pigs. The anti-CDV antibody plasma is hydrolyzed by pepsin. The second channel is the product of the above hydrolyzed plasma which is separated and purified by cation exchange resin and then extracted with 0.2 M NaCl, and the third channel is separated and purified by cation exchange resin to 0.5. The product eluted by M NaCl. All three measured the signal at 100 kDa, which is equivalent to the molecular weight of the expected IgG antibody F(ab') ₂ fragment.

Further calculating the recovery rate of the F(ab') ₂ fragment product, which is obtained by using 1000 ml of plasma with a neutralization price of 2700 times before purification, and the above F(ab') ₂ fragment production process can be obtained. The 380 ml, protein concentration 7.9 mg/ml, and the F(ab') ₂ product with a neutralization price greater than 6400 times, the recovery rate is greater than 90% at the neutralization price.

Example 2: Cation exchange resin purification method for samples with different antibody concentrations

Pigs have different immune responses due to individual differences and feeding environment. Taking CDV-infected pigs as an example, samples of 3 batches of immune serum were analyzed by SDS-PAGE and Comass blue (commassive brilliant blue) ) Dyeing, the results of which are shown in Figure 4. Although the total protein content of the injection analysis was equal, it was observed that the content of porcine serum albumin (PSA) was not much different, but the content of antibody (IgG) was greatly different. However, the preparation method of the present invention can be applied to samples of different concentrations, as shown in Fig. 5, the immunized pig serum and the product purified by the method of the present invention are analyzed by SDS-PAGE and coomass blue (commassive brilliant) Blue) staining, in which the ratio of IgG content shown in the first lane is much higher than the ratio of IgG content shown in the first lane of Fig. 2, but in the high concentration IgG serum (plasma) sample, the antibody F(ab') The recovery of ₂ fragments can still be greater than 90%.

Example 3: Neutralizing Antibody Price Test

The F(ab') ₂ fragment product was subjected to serial dilution and subjected to neutralization antibody titer test. No cell lesion was observed in the diluted 6400-fold sample, and cytopathic effect was observed in the 12800-fold diluted sample. The neutralizing antibody price was set at 6400 times.

The present invention may be applied to its broadest scope without further elaboration, based on the description of the present invention. Therefore, it is to be understood that the claims and claims are not intended to limit the scope of the invention.

Figure 1 shows a chromatogram of the separation and purification of antibody F(ab') ₂ fragments with a cation exchange resin, wherein the unlabeled peaks represent substances that are not bound to the column, and then 0.2 M and 1.0 NaCl, respectively. And 0.1 M NaOH flushing column. .

Figure 2 is a graph showing the results of SDS-PAGE analysis of products at various stages in the preparation of antibody F(ab') ₂ fragments and staining with comass blue. M : molecular weight marker; 1 : unhydrolyzed and purified plasma containing porcine anti-CDV antibody, wherein PSA represents porcine serum albumin, IgG antibody has a molecular weight of about 135 kDa; 2 : product of the aforementioned plasma by pepsin hydrolysis; 3 : The hydrolyzate was subjected to dialysis and displacement of the buffer product through a 50 kDa ultrafiltration membrane. There was no difference in protein composition distribution before dialysis using an ultrafiltration membrane, indicating that the ultrafiltration membrane dialysis could not obtain the effect of separation and purification; 4 : Filter of the cation exchange column; 5 : The cation exchange column was washed with 0.2 M NaCl to elute the column, and the isolated and purified antibody F(ab') ₂ fragment was observed.

Figure 3 shows the presence and degree of purification of antibody F(ab') ₂ fragments by Western blot analysis. Here, a rabbit anti-porcine IgG F(ab') ₂ fragment antibody (NOVUS) was used as a primary antibody for detection. 1 : a product containing pepsin hydrolyzed by a plasma containing a porcine anti-CDV antibody; 2 : a product of the above-mentioned hydrolyzed plasma purified by a cation exchange resin and 0.2 M NaCl; 3 : after the above-mentioned hydrolyzed plasma is purified by a cation exchange resin The product was eluted with 0.5 M NaCl.

Figure 4 shows the results of serum sample analysis of different batches of pigs infected with CDV. Serum samples with equal total protein content were analyzed by SDS-PAGE and stained with comassive brilliant blue. M : molecular weight marker; 1, 2, 3 : serum samples from different batches of pigs.

Figure 5 shows that the preparation method of the antibody F(ab') ₂ fragment of the present invention can be applied to serum samples of high concentration IgG. The immunized pig serum and the purified product were analyzed by SDS-PAGE and stained with comassive brilliant blue. M : molecular weight marker; 1 : unhydrolyzed and purified plasma containing porcine anti-CDV antibody, wherein PSA represents porcine serum albumin, IgG antibody has a molecular weight of about 135 kDa; 2 : the aforementioned plasma is subjected to pepsin hydrolysis and cation exchange resin The product after purification. The recovery of antibody F(ab') ₂ fragments can still be greater than 90% in high concentration IgG serum (plasma) samples.

Claims (7)

A method of preparing an antibody F(ab') ₂ fragment, comprising the steps of: hydrolyzing an antibody with pepsin to obtain an antibody F(ab') ₂ fragment; and the antibody F(ab') ₂ fragment Concentrating and replacing a buffer with a filtration system wherein the replacement of the buffer causes the F(ab') ₂ fragment of the antibody to have a positive charge; and the F(ab') ₂ fragment of the antibody is subjected to a cation exchange column Purification and replacement of a purified antibody F(ab') ₂ fragment with a salt aqueous solution, wherein the purified antibody F(ab') ₂ fragment does not contain serum albumin, intact antibody molecule and antibody Fc fragment, and wherein The filtration system is a Tangential Flow Filtration System. The method of claim 1, further comprising the step of concentrating the purified antibody F(ab') ₂ fragment in a second filtration system and replacing a second buffer. The method of claim 1, wherein the pH of the buffer is less than the isoelectric point (pI) value of the F(ab') ₂ fragment of the antibody. The method of claim 4, wherein the buffer is a sodium acetate solution having a pH of 4.3. The method of claim 1, wherein the aqueous salt solution is an aqueous solution of sodium chloride (NaCl) having a concentration of from about 0.1 M to about 2 M. The method of claim 2, wherein the second buffer is physiological saline. The method of claim 1, wherein the anti-system is IgG, IgA or IgM.