AU1331892A - Method of producing f(ab')2 fragments of immunoglobulins - Google Patents

Description

METHOD OF PRODUCING F(ab') FRAGMENTS OF IMMUNOGLOBULINS

2

Technical Field

The invention is directed to the production of F(ab') and Fab' fragments from immunoglobulin proteins by digestion of the proteins in acidified culture medium. The invention is particularly related to the producton of F(ab' ) fragments from monoclonal antibodies by acidification of the supernatant containing such anti¬ bodies after cultivation of hybridoma cells in a culture medium. Background Art

Immunoglobulins of the G class (IgG) consist of two heavy chains and two light chains. The light chains are each joined to a heavy chain by a disulfide (S-S) bridge and the two heavy chains are joined to each other by one to three disulfide bridges, depending on the subclass of the immunoglobulin (IgGl, IgG2a, IgG2b or IgG3), at a point known as the hinge region. Approximately one half of each heavy chain is intertwined with a light chain to form the antigen recognition structure of the immuno¬ globulin and the other half of each of the two heavy chains interact tightly by non-covalent forces. Diges¬ tion of an IgG molecule leads to the formation of various fragments. An F(ab') fragment results when the IgG molecule is split such that the two intertwined light- heavy chain portions of the IgG molecule are still linked by one or more S-S bridges at the hinge region. (E.g., pepsin cleavage). The non-covalently interacting heavy chain portion of the IgG molecule splits off to form a fragment designated Fc. The S-S bridging group or groups in an F(ab') fragment can be subsequently cleaved by chemical reduction to form two Fab' fragments. Fab frag¬ ments can be formed directly from an IgG molecule by cleaving the molecule such that the S-S bridge is cleaved or is carried with the Fc fragment. (e.g., papain cleavage). In subsequent discussions, the Fab and Fab' fragments will be treated as similar entities with regard to antigen binding and pharmacokinetics. (See Fig. 6, Diagram of an IgG Molecule, with some extraneous matter deleted, taken from Dictionary of Immunology, Third Edition, eds. W.J. Herbert et al. (London: Blackwell Scientific Publications, 1985), page 108). Fab and Fab' fragments formally differ as to their route of formation and the possibility as to whether any portion of the hinge region is attached to the fragment. Fab fragments are formed by cleavage such that no part of the hinge region is carried with the Fab fragments. Fab' fragments are formed by chemically cleaving F(ab') by reduction of the disulfide bond(s) and may or may not retain a portion of the hinge region. Immunologically, Fab and Fab' frag- ments react in essentially the same manner. Immuno¬ globulins other than IgG also give rise to F(ab') , Fab' (Fab) and Fc species and are meant to be included within the scope of this invention. IgG is thus used as the model immunoglobulin because it is the most common immunoglobulin and generally produces the most stable fragments.

A F(ab') fragment has a molecular weight of approx¬ imately 100,000 daltons and is usually stable. The Fab' fragments resulting from chemical reduction cleavage of a F(ab') fragment (e.g. by cysteine) or Fab fragments formed directly from IgG have molecular weights of approximately 50,000 daltons. The Fc portion of the IgG molecule can either form a stable fragment (Fd) or it may be further cleaved into numerous small fragments. Diges- tion of an IgG molecule with the proteinase pepsin typically results in the formation of F(ab') and Fc fragments, and digestion with the proteinase papain typically results in the formation of Fab and Fc frag¬ ments. F(ab') and Fab' fragments of monoclonal antibodies are useful in both diagnostic and pharmaceutical applica¬ tions because they represent smaller fragments of the IgG molecule which retain the antigen specific binding properties of the parent without the undesirable side ef¬ fects of the parent that are attributed to the Fc por¬ tion. Both F(ab') and Fab' fragments are useful in techniques such as radioimmunoscintigraphy and radio- immunotherapy because each has different pharmacokinetic and excretion characteristics and the absence of the Fc moiety decreases undesirable non-specific binding. At the present time, however, these fragments have not been exploited as fully as they might be because of the dif¬ ficulties encountered in their manufacture.

For in vivo application, the smaller F(ab') and Fab' fragments of the immunoglobulins distribute them¬ selves in the extravascular volume of the patient much more rapidly than the intact parent immunoglobulin distributes itself. This allows the F(ab') and Fab'

2 fragments to react with extravascular antigen more rapidly and thus speed the occurance of the desired ef¬ fect in the patient. Furthermore, the smaller size of the fragments allows them to be cleared from the patient's body more rapidly than the larger parent im¬ munoglobulin. This rapid clearance allows the person overseeing treatment to modify the kinetics of a mono¬ clonal antibody based radioactivity or drug delivery sys- tern for more rapid delivery, thus effectuating, in es¬ sence, a more rapid treatment of the patient.

The human anti-mouse immune response that is mounted in response to the in vivo administration of mouse immunoglobulin can be reduced by administrating the F(ab') and Fab' fragments. This is, in large part, due to the removal of the Fc portion of the immunoglobulin that is the principle immunogenic site of the immuno¬ globulin. The Fc portion of the immunoglobulin is also responsible for undesirable interactions with Fc recep- tors in several tissues which in turn are in part respon¬ sible for undesirable accumulation of monoclonal immuno¬ globulin. The tailoring of treatment to individual patients by using a mixture or "cocktail" of more than one monoclonal antibody is also enhanced by the use of antibody frag¬ ments. For example, because the F(ab') fragment of im- munoglobulins is held together by easily reduced disul¬ fide bridges, it is possible to produce hetero-bifunc- tional molecules by covalently linking the F(ab') frag¬ ments (one-half of an F(ab') ) of two monoclonal anti-

2 bodies of differing specificities using a method similar to that described by Brennan et al. in Science, 229: 81 (1985). For in vivo tests, the Fc mediated binding of immunoglobulin to cells such as macrophages and monocytes can be greatly reduced by using immunoglobulin fragments, thus reducing the background level of such assays. Since the F(ab') and Fab' fragments retain the antigen binding characteristics of the intact parent immunoglobulin with¬ out the undesirable side effects that are attributed to the Fc portion which is split off from the parent during digestion, the manufacture and use of these fragments is highly desirable. In the method of converting immuno¬ globulins to fragments described in the present inven¬ tion, technically simple procedures allow for the pro¬ duction of pharmaceutical or diagnostic grade immuno¬ globulin fragments in high yield. A major problem in the manufacture of F(ab') and

Fab¹ fragments is that the yield of the fragments is gen¬ erally low, less than 50%. Presently, production pro¬ cedures for highly purified IgG fragments require that the IgG be purified from ascites fluid or cell culture medium before the digestion procedure can be initiated. These procedures also require the addition of a purified protease such as chymopapain, trypsin or pepsin to the immunoglobulin in order to accomplish the digestion. Both of these requirements add to the manufacturing cost and complicate quality assurance. Pharmaceutical reagents must be low in endotoxin content and must be sterile. The proteases which are added to immunoglobulin must be specially purified under the same conditions and to the same standards as pharmaceuticals in order to to be useful for the production of pharmaceutical grade monoclonal antibodies. In addition, extraneous products

5 arising from use of the protease must be removed. Alter¬ nately, the proteases may be affixed to a support which is separable from the monoclonal antibody fragments pro¬ duced by the protease. Supported proteases have the ad¬ ditional problem that there is considerable instability

10 among some of the supported proteases used to produce IgG fragments. This instability can lead to contamination of the fragments produced by supported protease breakdown products. Lastly, digestion reactions involving sup¬ ported reagents, such as chymopapain conjugated to beaded

15 agarose, must be tested frequently or the digestion reac¬ tion will fail due to incomplete digestion or over-diges¬ tion of the immunoglobulin.

Watanabe et al., Vox Sang., 48: 1-7 (1985) have de¬ scribed a procedure for producing Fab and Fc fragments 0 from human immunoglobulin G by treatments with the plasma derived protease plasmin. Parham, J. Immunol., 131: 2895-2902 (1983) has reported producing Fab and or F(ab') fragments of mouse immunoglobulins by treatment

*£- with pepsin, chymopapain or trypsin. Parham demonstrated 5 that relatively pure (>50%) F(ab') fragments of mono- clonal antibodies could be produced directly from ascites fluid by using pepsin after lowering the pH of the as¬ cites fluid to 3.5 by the addition of citric acid. (Maximum pepsin activity occurs about pH 2.0, but usually u leads to complete breakup of the immunoglobulin into use¬ less fragments). The contaminating serum proteins in as¬ cites fluid were digested to mainly low molecular weight fragments that were eliminated by dialysis and the

F(ab') fragments were purified by chromatography of the 2 reaction mixture on diethylaminoethyl (DEAE) cellulose using Tris-HC_l [tris(hydroxymethy_lamino)methane-HCl] , _PH 7.5 as eluant. U.S. Patent No. 4,937,183 ('183 patent) to Ultee et al. describes a method for the preparation of antibody fragments by digestion of an antibody-compound or an antibody containing imtermediate compound conjugate using pre-activated thiol proteases such as papain, chymopapain and ficin. The F(ab') fragments resulting from practice of the '183 patent do not exist as free F(ab' ) fragments, but rather exist as part of an anti- body F(ab' ) -compound, intermediate or conjugate. Schlaeger et al.. Develop. Biol. Standard, 66: 403-408 (1987), describe a method of producing F(ab') fragments

__u from serum-reduced or serum-free culture supernatants that have been acidified to a pH less than 4.5 and in¬ cubated for 16 hours at 37° C. Schleager et al. cultured hybridoma cells in culture media containing 10%, 5%, 2.5%, 0.5% and 0% fetal calf serum (FCS). After cultur¬ ing the hybridoma cells and separating them from the supernatant, the supernatant was acidified and incubated. No protease such papain or pepsin was added to the acidified supernatant. Schlaeger et al. found that when a low level of FCS (0.5% or 0%) was used in the culture medium, significant protease activity resulted after acidification and incubation. At higher FCS levels, the protease activity of the acidified supernatant was reduced. These authors speculated that the protease ac- tivity results from the presence of possibly one or two cellular (acid) proteases similar to the lysosomal cat- hepsin D present in the medium after conditioning by the hybridoma cells. That is, the proteases arise and the medium is conditioned by the culturing of hybridoma cells in the medium. Schlaeger et al. found that the protease activity in the acidified supernatant was 100% inhibited in the presence of pepstatin A.

While the prior art has described the production of F(ab') and Fab fragments by digestion of G class immuno- globulin with and without the addition of a protease, none of that art describes a method commercially useful for the production of large quantities of F(ab') or Fab fragments. The known art, alone or in combination, de¬ scribes methods which are lengthy, costly and sometimes uncertain in the quality and purity of their product. F(ab' ) fragments are available, but their use in clinical studies is quite limited and they are generally expensive relative to IgG. This "economic unavailabil¬ ity' of F(ab') and Fab fragments, despite the fact that their utility is widely recognized, indicates that there is an unfulfilled need for a method of economically pro- ducing such fragments. The present invention describes a process for the production of such fragments in an eco¬ nomical manner by the addition of an acidifying compound of acceptable pharmacological presence to a hybridoma conditioned supernatant fluid containing a monoclonal antibody.

Discloure of Invention

The invention relates to a method of producing anti¬ body F(ab') and Fab' fragments by digesting immunoglobu¬ lin protein present in substantially serum free tissue culture medium conditioned by hybridoma cells which gave rise to the immunoglobulin. The method requires the ad¬ dition of a pharmaceutically acceptable acid to the cul¬ ture medium (for diagnostic applications, a wider range of acids could be used), digesting the immunoglobulin in the culture medium at a digestion temperature in the range of 15-50° C for sufficient time to convert the immunoglobulin to F(ab') and/or Fab' (by reductive

*£» chemical cleavage after neutralization) fragments without converting said immunoglobulin into useless fragments, neutralizing the digestion medium after the digestion is essentially complete, and filtering the fragment contain¬ ing solution to remove undesirable components. (Conversion of IgG to fragments has been accomplished at 4° C, but the time required is longer. However, should the nature of the immunoglobulin and/or fragments require lower temperatures, this can be accomplished according to the present invention). The invention describes a method for producing F(ab' ) and Fab' fragments free of undesir¬ able Fc fragments, endotoxins and other substances whose presence would prevent pharmacological use of such frag¬ ments or require expensive purification procedures. The method of the invention uses citric acid to acidify a hy¬ bridoma culture supernatant to a pH in the range of 2.0-5.0. The use of citric acid is preferred over the use of protease for digesting immunoglobulins because of the ease with which citric acid/sodium carbonate solutions can be sterilized and because citric acid is generally not regarded as a contaminant. Alternative acids which can be used without being regarded as con¬ taminants are phosphoric, hydrochloric, acetic and the like. The product solutions of F(ab')2 and Fab' frag- ments which result from the claimed invention may contain citric acid and sodium carbonate which are not regarded as contaminants. Brief Description of Drawings

Fig. 1 illustrates two distinct protease activities from MY904 conditioned medium after HPLC DEAE chromatography;

Fig. 2 illustrates the two dimensional crossed im- munoelectrophoresis of reduced and unreduced MY904 F(ab') fragments prepared according to the method of the

__u invention;

Fig. 3 illustrates the SDS polyacrylamide tube gel electrophoresis (SDS-PAGE) of MY904 F(ab') fragments;

Fig. 4 illustrates the immunoelectrophoresis of one lot of MY904 monoclonal antibody and three lots of MY904 F(ab') fragments;

Fig. 5 illustrates the results of Ouchterlony tests with MY904 F(ab') using three different goat anti-sera, one anti-serum to each Ouchterlony plate; and

Fig. 6 illustrates the F(ab') , Fab, Fc and Fd por- tions of an IgG molecule.

Best Mode for Carrying Out the Invention

The present invention demonstrates the principle and critical effect of lowering the pH has on the enzymatic digestion of immunoglobulins. The effect is to make the immunoglobulin molecule susceptible to proteolytic diges¬ tion, whereas as at approximately neutral pH, immuno- globulins are notoriously difficult to digest. The prob¬ able mechanism is a denaturing (or an alteration of the tertiary structure) of the Fc domain of the immunoglobu¬ lin molecule, thus making it susceptible to protease substances that are present. The examples given below best illustrate the results achieved using the method of the invention.

Glossary of Terms.

Immunoglobulin is abbreviated Ig. As used herein, the terms monoclonal antibody, immunoglobulin, and immunoglobulin protein are used interchangably unless in¬ dicated otherwise.

F(ab') , Fab and Fc fragments arise from the diges- tion of immunoglobulins, monoclonal antibodies, or immunoglobulin proteins.

Fab' arises from chemical reduction cleavage of an F(ab') fragment and contains a portion of the disulfide hinge region.

Fab arises directly from cleavage of immunoglobulin and does not contain any portion of the disulfide hinge region.

Fab' and Fab fragments are immunologically equivalent with regard to antigen binding and pharmacokinetics. Best Mode of the Invention

The method of the present invention requires only the addition of citric acid to unpurified monoclonal an¬ tibody IgG containing hybridoma conditioned medium to ac¬ complish the digestion of IgG to F(ab') or Fab' frag- ments. All subclasses of IgG may be utilized in the met¬ hod of the invention. Solutions of citric acid and sodium carbonate can be sterilized and rendered non- pyrogenic with relative ease compared to protease solution. In the best mode of the method of the inven¬ tion for the production of F(ab') or Fab' fragments, 1M citric acid solution was added to hybridoma culture med- ium that contains an immunoglobulin such as a monoclonal antibody. The hybridoma cells used to produce the mono¬ clonal antibody were removed from the medium prior to the addition of the citric acid. Alternately, a commercially available instrument such as an ACCUSYST machine (ENDOTRONICS Corp. of Coon Rapids, Minnesota) can be used to produce a monoclonal antibody containing conditioned medium free of hydridoma cells. Sufficient citric acid was added to the conditioned medium to adjust the pH to an optimal value, generally pH 3.5, but pH 5.0 for mouse IgG monoclonal antibody. Typically, 0.04 ml of 1M cit- ric acid was added per milliliter of conditioned medium. After acidifying, the medium was incubated for a time in the range or about 1 hour to about 48 hours at a tempera¬ ture in the range of about 20° C to about 40° C. The op- timal time has generally been about 18 hours and the op¬ timal temperature has generally been about 25° C. The conversion of immunoglobulin to F(ab' ) fragments was stopped, when desired by such as the exhaustion of im- munoglobin starting material or the occurance of side reactions, by increasing the pH of the reaction solution through the addition of 1M sodium carbonate solution or similar basic solution. (The amount of 1M sodium car¬ bonate required is approximately 2.5 times the volume of citric acid added). Low molecular weight peptide frag- ments remaining after digestion were eliminated, and the salts and buffer of the fragment solution were simul¬ taneously changed to 5 mM potassium phosphate (pH 7.5), by diafiltration using a 30,000 nominal molecular weight cut-off membrane. The F(ab') product was eluted from the DEAE cellulose column while the parent immunoglobulin and other contaminants remain bound to the DEAE column. Typically, the only contaminant, if any, detectable by gel electrophoresis is less than 5% undigested immuno¬ globulin. The undigested immunoglobulin was reduced to less than 1% by chromatography on a sterile, low pyrogen column of DEAE cellulose. The method was applicable to all mouse immunoglobulin G subclasses IgGl and IgG3. At least some mouse IgG2 class antibodies resist this proce¬ dure. The Fab' fragments of immunoglobulins can be pro¬ duced from the purified F(ab') fragments produced by this method by chemical reduction using reducing agents such as dithiothreitol, cysteine or 2-mercaptoethanol. Any excess reducing reagent can be removed by diaflow, dialysis, or gel filtration chromatography. The Fab' fragments have not been observed being produced directly from the immunoglobulins tested to date. Highly purified F(ab') fragments of immunoglobulin were obtained in yields ranging from 84% to 104% of theoretical on a molar basis. (Table 9, greater the 50% on a mass basis). Mouse IgG monoclonal antibodies con¬ vert using milder conditions than those used for mouse IgGl antibodies. Generally, mouse IgG3 can be converted to F(ab') fragments by incubation, for example, at 37 °

C, pH 5.0 for about 3 hours.

The immunoglobulin MY904 was used for the experi¬ ments summarized in the Tables and Figures contained herein. The MY904 monoclonal antibody was selected for exemplary purposes only F(ab') fragments were sucessfully produced from a variety of other monoclonal antibodies which are available from hybridoma cell lines on deposit with the American Type Coulter Collection or from Coulter Corporation, the Assignee of the application. The other monoclonal antibodies are KC-4, IgG3 type, A.T.C.C. deposit No. HB8709; KC-4, IgM type, A.T.C.C. deposit No. HB8710; Control Mouse IgGl from Coulter Corporation; Til from Coulter Corporation; Mc5 from Coulter Corporation; BrE3, A.T.C.C. deposit No. HB10028; 2H4 from Coulter Corporation; and KC-16, A.T.C.C. deposit No. CRL8994. The MY904 and the other tested immunoglobulins as listed herein are mouse immunoglobulins. The claimed in¬ vention, however, is applicable to immunoglobulins from other species such as, for example, human, rabbit, goat or rat immunoglobulins. Further, chimerized immunoglobu¬ lins may be used in the claimed invention or fragments obtained using the invention may be chimerized.

In the production of F(ab' ) and/or Fab' fragments, it is important that the conditioned medium used as starting material does not contain high levels of non- immunoglobulin proteins, e.g. serum proteins, as these will cause contamination of the final product and inhibit the progress of the digestion reaction. The culture med¬ ium used in the present invention contains only a few widely known protein additives, such as insulin and epitheliar growth factor, and is serum free.

The correct pH, reached by citric acid addition, for optimizing the yield of each monoclonal antibody was ex¬ perimentally determined. The determination was made by adding sufficient citric acid to conditioned medium samples to lower the pH to 4.5, 4.25, 4.00, 3.75, 3.5, 3.25, and 3.00. Each acidified sample was then incubated for 18 hours at 25° C. The IgG present in each sample converts to F(ab') fragments at a rate that is dependent on pH. The percentage of IgG converted to F(ab' ) was determined by analytical gel filtration chromatography with a TSK-R-250 HPLC column (BioRad Corporation, Richmond, California) using phosphate buffered saline as solvent. The percentage of IgG vs. F(ab' ) is determined by the relative peak heights measuring absorbance at 280 nanometers (run). The pH dependence of the conversion of IgG to F(ab') (18 hrs., 25° C) are shown in Table 1. Table 1.Relationship between citric acid adjusted pH and the formation of F(ab') in hybridoma conditioned medium for monoclonal antibody MY904.

Sample Incubated mg/ml MY904 IgGl mg/ml MY904 F(ab')

The results shown in Table 1 indicate that pH 3.5 is optimal for the conversion of IgG to F(ab') . The mono¬ clonal antibody used in the experiments, MY904 (Coulter Corporation, Hialeah, Florida) is of IgGl isotype. The results of Table 1 are valid for all IgGl isotype mono¬ clonal antibodies. The results indicate a maximum yield of F(ab') and undeσtable amounts of immunoglobulin. Al-

__u though the optimum pH is 3.5, significant conversion of immunoglobulin occurs at pH 4.5 and higher.

The effect of temperature on the yield of F(ab') was examined in a similar series of experiments. MY904 hybridoma conditioned medium was adjusted to pH 3.5 using citric acid. The sample was then split into aliquots and incubated for 18 hours in water baths of varying temper¬ atures. The results are shown in Table 2. Table 2. Relationship between incubation temperature and yield for MY904 conditioned medium adjusted to pH 3.5 and incubated for 18 hours.

Incubation Temp. mg/ml MY904 IgGl mg/ml MY904 F(ab' ) Control, pH @ 7.4 0.62 None dectectable 4° C, pH = 3.5 0.29 0.14

As shown in Table 2, although the IgG concentration decreased as the temperature was increased, the optimal temperature for the reaction was found to be 25° C. A significant loss of F(ab' ) fragments occured at temper¬ atures above 25° C. Most likely, at higher temperatures the kinetics of further enzymatic degradation of the F(ab' ) in the pH 3.5 medium is accelerated relative to the digestion of Ig such that the F(ab' ) fragment is digested into smaller fragments more rapidly than it is formed.

The next series of experiments was undertaken to determine whether the digestion activity depended upon an active material produced by the hybridoma cells or whet¬ her digestion was dependent upon the antibody pH or temperature alone. Purified MY904 monoclonal antibody was added to Nutridoma-SP cell culture medium (Boehringer-Mannheim Corporation, Indianapolis, Indiana) at a concentration of about 0.5 mg/ml. The pH of the culture medium was adjusted to 3.5 using citric acid, the antibody containing medium split into several samples, and individual samples of medium were incubated for approximately 18 hours at various temperatures. Since previous experiments had shown that the rate of en¬ zymatic reaction increases with temperature, more rapid conversion of immunoglobulin to F(ab' ) fragments should occur at the more elevated temperatures. Consequently, one would predict a reasonable yield of the F(ab' ) frag- ments, with minimum IgG contamination, could be achieved by incubations at temperatures above 25° C (e.g., 37° C) for times less than the 18 hours usually employed in these experiments. The results of these experiments, shown in Table 3, demonstrate that there is some loss of IgG without formation of F(ab') as temperature is increased.

Table 3. Effect of between incubation temperature and yield of F(ab' ) for purified MY904 without con- ditioned medium (non-conditioned Neutrocyte added) adjusted to pH 3.5 and incubated for 18 hours

mg/ml MY904

None detectable None detectable None detectable None detectable

While the results in Table 3 show a steady decrease in MY904 antibody concentration with increasing temper¬ ature, no corresponding increase in F(ab') fragment con- centration appears in any of the test samples. The results indicate that the digestion of antibody to F(ab') fragments is dependent upon some substance which is or acts like a protease, and is co-produced by the hybridoma cells or arises from the hybridoma cells during antibody production. Isolation and purification of the monoclonal antibody results in the loss of the substance. Consequently, after isolation and purification, no di¬ gestion of immunoglobulin to F(ab') fragments upon ad-

*£» dition of citric acid occurs because the digesting substance or promoter is missing.

During experiments using monoclonal antibody MY904, what seems to be a large excess of proteolytic enzyme was found to be present in at least some lots of hybridoma conditioned medium. In order to determine whether there was sufficient proteolytic enzyme to convert monoclonal antibody in excess of the endogenous immunoglobulin, samples of conditioned medium containing 3 mg/ml MY904 monoclonal antibody (Original Medium, OM) were spiked with up to an additional 20 mg of purified MY904 antibody after the pH of the medium had been adjusted to 3.5 with citric acid. The spiked samples were incubated for 3 hours at 37° C and assayed. The results are shown in Table 4 and indicate that conditioned medium contains sufficient "protease" to digest additional, purified im- munoglobulin.

Table 4. Excess F(ab') forming activity when purified

* _*

MY904 monoclonal antibody is incubated in con¬ ditioned Nutridoma-SP cell culture medium at pH 3.5 for 3 hours at 37° C.

Theoretical yield (%) of

Incubation Sample MY904 F(ab' ) obtained

Original Medium (OM) 100%

OM + 1 mg/ml 100%

OM + 5 mg/ml 98.7% OM + 7.5 mg/ml 98.9%

OM + 10 mg/ml 98.9%

OM + 20 mg/ml 99.1%

OM = Original Medium = 3 mg/ml monoclonal antibody MY904.

Observed F(ab' )

* denotes 2 X 100%

0.67 X spiked IgG

In order to determine whether or not residual protease activity remains in the F(ab' ) product after diafiltration, bulk concentrates of five (5) MY904

F(ab' ) final products were spiked with purified MY904 immunoglobulin. Aliquots of the spiked products were tested as is (pH 7.2) or were adjusted to pH 3.5 and in¬ cubated for 18 hours at 25° C. The quantity of residual IgGl immunoglobulin was then determined by high pressure liquid chromatography (HPLC). No changes in the con- centration of F(ab') were observed. The results, Table

2 5, indicate that when the pH is adjusted to 3.5, 2% or less of the original immunoglobulin converting activity remains in the F(ab') final product. There are no con- taminants in the F(ab') product detectable by met- hodologies such as immunoelectrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), with or without reduction, or Ouchterlony immmunop- recipitation outside of the residual enzymatic activity observed.

Table 5. Depletion of protelytic activity in 5 lots of purified MY904 F(ab') product

Lot # F(ab') mg/ml spiked puri- product fied MY904 *c*onverted by product

* The Diaflow concentration over a 30,000 daltons nominal cut-off membrane and DEAE cellulose purification procedure used to purify the F(ab') products leaves only traces of immunoglobulin con¬ verting protease. ** Determined by spiking 20 mg/ml purified MY904 into the purified F(ab') product, incubating at pH 3.5 and determining the residual MY904 IgG by HPLC.

Characterization of the protelytic activity present in hybridoma conditioned medium.

Proteolytic enzymes can be categorized and identi¬ fied by the inhibitors that are effective and ineffective at inhibiting their activity. The four common types of proteases that encountered are: (1) serine active site; (2) thiol active site; (3) metal requiring active site proteases; and (4) the acid proteases related to the pep- sin or lysosomal cathepsins. In general, serine active site esterases (proteases included) such as trypsin and chymotrypsin are inhibited by active site alkylating com¬ pounds such as phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorylphosphate (DFP) or the peptide trypsin inhibitor from soybeans. The thiol proteases have a cys- teine active site that is sensitive to mercurials (thimerosal) and to PMSF. The metalloproteases are in¬ hibited by metal coordination compounds such as ethylene- diamine tetraacetic acid (EDTA). Only acid proteases, such as pepsin and cathepsin-D are generally inhibited by a specific peptide inhibitor, pepstatin A. To determine the type of protease in hybridoma conditioned medium that is responsible for the conversion of immunoglobulin to F(ab') and/or Fab' fragments, the production of F(ab') from MY904 conditioned medium was monitored. In this ex¬ periment, each inhibitor to be tested was added to an aliquot of the MY904 (3 mg/ml) conditioned medium. The pH of the conditioned medium was adjusted to 3.5 with citric acid and the conditioned medium was incubated for 18 hours at 25° C. An aliquot of the conditioned medium which did not contain inhibitor was adjusted to pH 3.5 and used as the control for the rate of conversion of MY904 immunoglobulin to its F(ab') fragment. Conversion was determined by chromatographic analysis where the peak height of the F(ab') or MY904 immunoglobulin was

*%-_* measured. An aliquot of the conditioned medium, without treatment of any type, was used as the control for the starting MY904 concentration. The results of this exper¬ iment to determine the specificity of the protease res- ponsible for the conversion of IgG to F(ab') are shown in Table 6. Table 6. Effect of protease inhibitors on the conversion of immunoglobulin to F(ab')

Substance/Treatment Percent of starting Percent of of conditioned IgG remaining at control

F(ab' )

2 ** medium at 18 hr. at 18 hr

relative untreated

** relative to pH 3.5 1 = pH 3.5 treated; 2 = 100 #g; 3 = 10 /g; and 4 = 100 mlu

Table 7 shows the results of experiments with puri¬ fied immunoglobulin spiked Nutridoma medium (not hybri¬ doma conditioned) with the purified protease cathepsin-D added. Otherwise, the experiment in Table 7 is similar to that in Table 6 where hybridoma conditioned medium was tested, except for the added cathepsin-D

The results shown in Table 7 indicate that cathepsin-D is capable of digesting mouse immunoglobulin to F(ab') . However, if cathepsin-D were the protease in

2 hybridoma conditioned medium responsible for the diges¬ tion of immunoglobulin, one would expect that the pattern of inhibition of hydrolysis of the cathepsin-D substrate would be the same as the pattern of inhibition of conver- sion of immunoglobulin to F(ab') that was observed in

Table 6. Since the pattern of inhibitors in Tables 6 and

7 are qualitatively similar, one can conclude that cathepsin-D is capable of converting immunoglobulin in hybridoma conditioned medium to F(ab') . Other

2 substances present in conditioned medium may influence the efficiency of the digestion or be the principal ac¬ tive agent.

Table 7. Effect of protease inhibitors on the conversion of MY904 immunoglobulin to F(ab') by cathepsin-D

Treatment of 3 mg/ml % of Starting % Control F(ab' )

* 2 MY904 immunoglobulin immunoglobulin at 18 hr. plus 0.5 U remaining at

# No cathepsin * Relative to pH 3.5

** Relative to untreated

1 = pH 3.5 treated; 2 = 100 g; 3 ■= 10 /g; and 4 = 10 ng.

Table 8 illustrates that the pattern of inhibition of hydrolysis of a broad spectrum protease substrate S- 2288 (H-D-Isoleucyl-L-propyl-arginyl-p-nitroanilide dihydrochloride, KabiVitrum, Stockholm, Sweden). The pattern of activity inhibition of cathepsin-D on the substrate S-2288 is quite different from the pattern of inhibition that was observed in Table 7. The conditioned medium of Table 8, before the addition of citric acid, has a pH greater than 7.4. The results shown indicate that the S-2288 substrate of cathepsin-D is degraded faster at pH 7.4 that at pH 3.5 (1.02 v. 0.5). This is contrary to earlier observations that the rate of conver¬ sion of mouse immunoglobulin G to fragments is much greater at acidic pH (pH 4.5 or below) than at neutral pH. This unexpected difference does not preclude the possibility that the S-2288 substrate which is acted on defines the active principle that is degrading the im¬ munoglobulin to F(ab') fragments. The significant ef- feet of lowering the pH to 4.5 or below may result in a structural or conformational change of the immunoglobulin which allows a protease access to an immunoglobulin se¬ quence that is highly resistant to proteolytic digestion at pH 7.4. Normally, immunoglobulins are highly resis- tant to digestion, in part because the molecule is so en¬ folded that reactive sites are protected from attack by cleaving reagents.

The results in Table 8 indicate that DFP is the only protease inhibitor that is effective at inhibiting the degradation of S-2288. Table 7 indicates that pepstatin, azide and thimerosal were the only species which sig¬ nificantly inhibited the conversion of immunoglobulin to F(ab') . One further observation is that the enzymatic activity measured by S-2288 degrades rapidly during in- cubation at pH 3.5 and 37° C. After 3 hours, the deg¬ radation of protease activity is so complete that there is almost no protease activity remaining that will deg¬ rade S-2288. This indicates that there will be little protease activity remaining in the F(ab' ) product and that the proteolysis is self-limited by autolysis of the protease at low pH. The IgG converting activity [to

F(ab') ] has been found by the inventor to be stable for 2 years at neutral pH.

Table 8. Effect of protease inhibitors on the ability of conditioned medium to cleave S-2288

1 rotease substrate.

1 = D-isoleucyl-L-Prolyl-L-arginine-p-nitroanilide.

2 = Units are: (picomoles nitroanilide liberated)/min./mL.

Fig. 1 resolves two distinct protease activities from MY904 conditioned medium using high pressure liquid chromatography (HPLC) with a diethylaminoethyl cellulose column (DEAE). Prior to chromatography, MY904 con¬ ditioned medium was treated with protein-A Sepharose to remove MY904 immunoglobulin. The protein-A column eluant was pooled and concentrated 10-fold over a YM-30 membrane as the salt. The buffer was exchanged for 0.01 M potas¬ sium phosphate, pH 7.2. The concentrated conditioned medium, minus immunoglobulin, was loaded into a protein pack 5PW DEAE HPLC column_, (Waters Corporation, Milford, Maine). A 0.01 M potassium phosphate solution was used to develop the column until baseline absorbance at 280 nanometers was attained. The column was then developed with a 0.1 M to 0.5 M linear gradient concentrations of potassium phosphate, pH 7.2. The potassium phosphate solutions were pumped onto the column and fractions of the column eluant were assayed for their ability to con- vert MY904 immunoglobulin to F(ab') , after spiking with MY904 IgG and acidification to pH 3.5, and for their S- 2288 cleaving activity.

The results, as shown in Fig.l, indicate that no sample with significant protease activity eluted either before the gradient was started nor after elution with

0.25 M potassium phosphate. The peak of S-2288 activity (indicative of serine protease) eluted significantly shifted from the two major peaks (A and B) of immunoglobulin converting to F(ab') activity that were observed. The two peaks (A and B) indicative of immuno¬ globulin converting activity were further characterized as to their sensitivities to protease inhibitors. Peak A was found to be inhibited by pepstatin, but not by diisopropylfluorylphosphate (similar to acid proteases like cathepsin-D or pepsin). Peak B was found to be in¬ hibited by diisopropylfluorylphosphate, but not by pepstatin (similar to serine or thiol proteases). These results clearly demonstrate that there are at least two (2) distinct active agents present in hybridoma con- ditioned medium that are capable of converting immuno¬ globulin to F(ab') fragments at acidic pH.

Table 9 shows the yield of MY904 F(ab') fragments

-_b from seven consecutive fragment preparations. Starting material was MY904 mouse IgG conditioned media. The starting quantitiy of antibody in each preparation was determined by a radioimmunoassay specific for the My904 monoclonal antibody. The theoretical yield of MY904

F(ab') was calculated to be 66.7% of the starting MY904

2 monoclonal antibody on a- mass basis [(100,000 daltons)/(150,000 daltons) = 0.667]. The percent yield was calculated as the actual yield in milligrams (mg) of

MY904 F(ab') as measured by Lowry protein divided by the 2 theoretical yield in milligrams. The mean yield of MY904

F(ab') over the seven productions runs was 95% of the theoretical yield.

Table 9. Yield of MY904 F(ab') from MY904 conditioned

2 medium from seven consecutive sam le re arations

Footnotes:

1. Starting quantity of MY904 monoclonal antibody, mg.

2. Theoretical yield of MY904 F(ab' ) fragments, mg.

3. Yield of MY904 F(ab' ) fragments, mg.

4. Percent yield of F(ab') fragments corrected for loss of Fc.

Table 10 shows the concentration of endotoxins, es¬ timated by the Limulus Amebocyte Lysate assay, for the MY904 conditioned medium starting material and the purified MY904 F(ab') final product for the seven con¬ secutive sample preparations described in Table 9. Total endotoxin content of the starting material was determined in the five preparations 3-7. In four of these five preparations (3, 4, 6 and 7), there was a decrease in the total endotoxin units from starting material to final product. In all seven of the preparations, product F(ab') had less than 2.5 endotoxin units per milligram 2 of F(ab') . Consequently, F(ab') doses of 2 mg/kg body 2 2 weight per hour, or higher doses, could be infused into a human under Food and Drug Administration (FDA) guidelines. FDA guidelines set the permissible endotoxin level at 5 endotoxin units/Kg body weight/hr.

Table 10. Concentration of endotoxins in MY904 conditioned starting material and purified MY904 F(ab') roduct of the sam le re arations of Table 9

Footnotes:

1. Total endotoxin units in MY904 conditioned medium

2. Total endotoxin units in MY904 F(ab') final pro-

2 duct.

3. Endotoxin units per milligram of MY904 F(ab') .

4. Not Determined.

Figures 2-5 graphically display the results of vari¬ ous tests with MY904 monoclonal antibody and F(ab')

2 fragments derived from MY904 monoclonal antibody.

Fig. 2 illustrates the results of two-dimensional immunoelectrophoresis of MY904 F(ab') fragments prepared

-_b as described above. Fifteen (15) micrograms of MY904

F(ab') was run in the first dimension (A) containing 1%

2 w/v agarose with 0.073 M Tris [tris(hydroxymethylamino- methane)], 0.024 M Barbital, pH 8.6 buffer. The second dimension of the gel (B) was then cast using 1% w/v agarose with 0.073 M Tris, 0.024 M Barbital, pH 8.6 buffer containing 1% v/v Goat anti-mouse whole serum

(Cappel). The plate was then turned 90 degrees and electrophoresed in dimension B. The gel was then washed and stained with Coomassie Blue R-250. The only precipitin arc identified as mouse F(ab') based upon its

2 electrophoretic mobility compared to a specific anti- ouse IgG serum control was precipitin arc (1). The conclusion of this analysis is that the lot of My904

F(ab') tested contains only mouse F(ab') and no other 2 2 detectable mouse proteins.

Fig.3 represents the results obtained from the SDS

[sodium dodecyl sulfate] polyaery1amide tube gel electro- phoresis (SDS-PAGE) of MY904 F(ab') . Reduced or un-

2 reduced 10 microgram {μg) samples of MY904 F(ab') was electrophoresed into polyacrylamide tube gels containing SDS. The unreduced separation gels (1 and 3) contained 5% acrylamide and 0.13% bis-acrylamide (Bis); while the reduced (by 2-mercaptoethanol) gels (2 and 4) contained 10 % acrylamide and 0.26% Bis. The separation gels con- tained final concentrations of 0.375 Tris-HCl, pH 8.8, with 0.1% SDS buffer. The stacking gels were 3.5% ac¬ rylamide and 0.09% Bis containing 0.125 M Tris-HCl, pH 6.8, with 0.1% SDS buffer. The electrode buffer con¬ tained 0.05 M Tris and 0.384 M glycine, pH 6.8, with 2% SDS, 20% glycerol and 0.005% Bromophenol Blue (plus 5% 2- mercaptoethanol for reduced sample gels). Staining was done with Coomassie Blue R-250.

Gel #1 is 10 μg of unreduced MY904 F(ab') electro¬ phoresed using a 5% acrylamide gel. A single band was observed with a molecular weight estimated at 97,500 dal¬ tons [similar to mouse IgG F(ab' ) ] by interpolation from the molecular weights versus the mobilities of the stand¬ ard proteins in Gel #3.

Gel #2 is 10 μg of reduced MY904 F(ab') electro-

2 phoresed using 10% acrylamide gel. One band was observed

(with some shadowing) with a molecular weight estimated at 25,500 (similar to the light chain of mouse IgG and the fragment of the heavy chain of mouse IgG after removal of the Fc). The molecular weight of this peak was estimated by interpolation from the molecular weight versus mobilities of the standard proteins of Gel #4. Gel #3 is the molecular weight standards: myosin

(200,000 daltons), beta-galactosidase (116,000 daltons), phosphorylase B (92,500 daltons), bovine serum albumin (66,000 daltons) and ovalbumin (43,000 daltons) electro¬ phoresed on a 5% acrylamide gel without reduction. ° Gel #4 is the molecular wight standards: beta- galactosidase (116,000 daltons), phosphorylase B (92,500 daltons), bovine serum albumin (66,000 daltons), oval¬ bumin (43,000 daltons), carbonic anhydrase (30,000 daltons) and trypsin inhibitor (20,100 daltons) electrop- horesed on a 10% acrylamide gel with reduction.

Analysis of the data of Fig. 3 shows that MY904 F(ab') , under non-reducing conditions, has a molecular

.__u weight of 97,500 daltons. This is consistent with the molecular weight of a mouse IgG F(ab') which is about 100,000 daltons. The analysis of the MY904 F(ab' ) under reducing conditions indicates that it contains disulfide linked sub-units of 25,500 daltons. This is consistent with a protein composed of four (4) sub-units of about 25,000 daltons and is similar to mouse IgG F(ab') which has two light chains and two half heavy chains, each of which is about 25,000 daltons. Analysis of MY904 IgG under similar non-reducing conditions gives a molecular weight estimate of 144,000 daltons. Under reducing con¬ ditions, MY904 IgG exhibits two bands which appear at 54,800 daltons (similar to mouse IgG heavy chain) and at 24,100 daltons (similar to mouse IgG light chain).

Fig. 4 represents three (3) lots of F(ab') product made from MY904 conditioned medium and one (1) lot of MY904 IgG by immunoelectrophoresis. Well 1 contains the MY904 monoclonal antibody. Wells 2 and 5 contain MY904

F(ab') fragments designated, for reference purposes, as

2 being Lot A fragments. Wells 3 and 6 contain MY904 F(ab' ) fragments similarly designated as Lot B frag¬ ments. Lastly, wells 4 and 7 contain MY904 F(ab')

2 designated as being Lot C fragments. Troughs A and C contain goat anti-mouse IgG serum specific for the Fc region of mouse IgG. Troughs B and D contain goat anti- mouse IgG serum specific for the F(ab') region of mouse IgG. Troughs E and F contain goat anti-mouse IgG serum that reacts with both the heavy and the light chain of mouse IgG. The MY904 and MY904 F(ab') samples were electro¬ phoresed in the gel with the positive and negative electrodes in the orientation shown in Fig. 4. After electrophoresis, goat anti-mouse sera was added to the troughs as indicated. Precipitin reactions were allowed. Non-precipitin protein was allowed to diffuse out of the gel before the gel was stained with Coomassie Blue to reveal the precipitin lines. In a given sample well, a precipitin arc moving away from the adjacent anti-serum indicates recognition of the sample by the anti-serum. The results for well 1, using goat anti-mouse IgG serum specific for the Fc region of mouse IgG, indicate the presence of Fc in the MY904 IgG. This is a positive control. Sample wells 2, 3 and 4 do not show a precipitin arc with troughs A and C which contain goat anti-mouse IgG serum specific for the Fc region of mouse

IgG. This indicates that there is no Fc present in these samples. This is consistent with the structure of

F(ab') , where Fc has been removed. Samples 2, 3 and 4

2 do show precipitin arcs with troughs B and D, thus in- dicating that the F(ab' ) structure is present. Sample

2 wells 5, 6 and 7 show precipitin arcs with the goat anti- mouse IgG serum that reacts with both the heavy chain and the light chain of mouse IgG. This reaction confirms that the F(ab') structure is present. Fig. 5 represents the results of Ouchterlony tests of MY904 F(ab') samples using three different goat anti-

2 sera, one anti-serum to each Ouchterlony plate. In these tests, MY904 IgG was loaded on the top well, well 1, of each of three Ouchterlony plates. Five different samples of MY904 F(ab') , Lots A'-E', were pipetted into the other five wells surrounding the anti-serum wells in the center of the each plate. A specific anti-sera was pipetted into the center well of the plate and diffusion was allowed to form a precipitin line. Non-precipitin proteins were washed out of the plates and the plates were stained for protein using Coomassis Blue. In clock- wise order from well 2 of each plate, the wells contained: well 2 - Lot A', well 3 - lot B', well 4 - Lot

C, well 5 - Lot D' and well 6 - Lot E'.

The center well of plate 1 was filled with goat anti-mouse IgG (intact IgG reacting with both light and heavy chains). The result of the Ouchterlony reactions of plate 1 was that all six samples formed a precipitin line indicating that all the samples contained mouse IgG.

The precipitin line from the sample of well 1 (MY904 IgG) showed incomplete identity with the two adjacent samples [2 and 6 which contain MY904 F(ab') ]. This is consis-

2 tent with MY904 having an Fc fragment that is lacking in the MY904 F(ab') samples. All five of the F(ab')

*£. mt samples in wells 2-6 showed complete identity with each other thus indicating that they are of very similar, if not the same, structure.

The center well of plate 2 was filled with goat anti-mouse IgG specific for F(ab' ) from mouse IgG. The results indicate that all six samples reacted with complete identity to each other. This indicates that each sample contains the F(ab' ) structure of mouse IgG. The MY904 IgG will react in the same manner as F(ab') fragments because although there is an Fc fragment in the MY904 IgG, there is no anti-serum titer to the Fc frag¬ ment. Consequently, there is nothing to cause an arc which indicates incomplete identity.

The center well of plate 3 was filled with goat anti-mouse IgG serum specific for the Fc region of mouse IgG. The only sample in wells 1-6 that showed a precipitin reaction reaction with the anti-serum was MY904 IgG in well 1. This indicates that mouse IgG Fc is present only in the intact MY904 IgG of of well 1 and is absent in the samples of MY904 F(ab' ) of well 2-6.

These results are consistent with and proof of the fact that the digestion and purification procedures described herein cleave and remove Fc from MY904 IgG. A priori, if the digestion and purification procedures worked, only well 1 would be expected to show a precipitin reaction.

Claims (24)

Claims

1. A method of producing antibody F(ab') and Fab'

-_U fragments comprising:

(a) acid digesting of immunoglobulin proteins pres¬ ent in substantially serum free tissue culture medium conditioned by hybridoma culture of said immunoglobulins;

(b) neutralizing the resulting acid digestion med¬ ium after the digestion is substantially complete; and

(c) isolating the F(ab') and Fab' fragments, said

__b

Fab' fragments being produced from said F(ab' ) fragments by the additional step, after (b), of reductive chemical clevage.

2. The method in accordance with claim 1 wherein citric acid is the acidifying acid.

3. The method in accordance with claim 1 wherein the acid digesting is at a pH between 2.0 and 5.0.

4. The method in accordance with claim 1 where wherein the acid digesting is at a pH of about 3.5

5. The method in accordance with claim 1 wherein the serum content of the conditioned medium is less than 5%.

6. The method in acordance with claim 1 wherein the conditioned medium is serum free.

7. The method in accordance with claim 1 wherein the immunoglobulins are selected from the group consist- ing of human, mouse, rabbit, goat and rat immunog¬ lobulins.

8. The method in accordance with claim 7 wherein the human immunoglobulins are produced in tissue cul¬ tures, by human-mouse tri-hybridomas or by transformed B- cells.

9. The method in accordance with claim 1 wherein the immunoglobulins are mouse immunoglobulins of the G class.

10. A method of producing F(ab') and Fab' frag- ments from mouse immunoglobulin proteins of class G which are present in tissue culture medium containing less than 5% serum and conditioned by hybridoma culture of said immunoglobulins, said method comprising adjusting the pH of the conditioned culture medium to between 2.0 to 5.0 by the addition of citric acid, and incubating the resulting citric acid and immunoglobulin containing med¬ ium at a temperature in the range of about 20° C to about 40° C to produce said fragments.

11. The method in accordance with claim 10 wherein the pH is about 3.5.

12. The method in accordance with claim 10 wherein the temperature is about 25° C.

13. The method in accordance with claim 10 wherein the immunoglobulin proteins are monoclonal antibodies.

14. A method of producing F(ab') and Fab' frag¬ ments from mouse class G immunoglobulin protein in substantially serum free tissue culture medium con¬ ditioned by hybridoma culture of said protein, said met¬ hod comprising: (a) separating the hybridoma culture medium from the hybridoma cells which were cultured;

(b) reducing the pH of the hybridoma cell free med¬ ium of step (a) to a pH in the range of about 3.0 to about 4.5 by the addition of an appropriate amount of citric acid ;

(c) incubating the acidified medium of step (b) at a temperature of about 25° C for a time sufficient to convert substantially all of the immunoglobulin protein to F(ab') and/or Fab' fragments; 2 (d) terminating the incubation of step (c) by the addition of a base to raise the pH of the medium to a pH in the range of about 6 to about 8;

(e) purifying the medium of step (d) by diafiltration using a meirtbrane with a nominal molecular weight cut-off of about 30,000 daltons to remove low molecular weight species and to change the salt and buff¬ er of the solution of (c) to 5mM potassium phosphate, pH about 7. 5 ; and

(f) chromatographing the solution of step (e), if necessary, to reduce its immunoglobulin content, to ob¬ tain a pharmacologicaly useful solution of monoclonal an- tibody F(ab') or Fab' fragments, said Fab' fragments being produced from said F(ab') fragments by the ad- ditional step, after step (d), of reductive chemical cleavage.

15. The method of claim 14 wherein the incubation time is in the range of 3 to 24 hours, the time being in¬ versely dependent on the pH and temperature.

16. The method in accordance with claim 14 wherein the serum is less than 5% of the medium.

17. The method in accordance with claim 14 wherein the medium is essentially serum free.

18. Monoclonal antibody F(ab') and Fab' fragments prepared by the method comprising acidifying a substan¬ tially serum free tissue culture medium conditioned by hybridoma culture and containing immunoglobulin proteins to a pH in the range of 2.0 to 5.0; incubating the acidified culture medium whereby said immunoglobulins are converted to said F(ab') ; adjusting the pH of F(ab') containing digestate medium to a pH in the range of 6 to 8; cleaving F(ab') fragment by chemical reduction if Fab' are the desired end product and purifying the pH ad¬ justed medium to obtain a solution of said antibody F(ab') or Fab' fragments.

19. The F(ab') or Fab' fragments of claim 18 where the fragments are derived from immunoglobulins selected from the group consisting of human, mouse, rat, goat and rabbit immunoglubulins.

20. The F(ab') or Fab' fragments of claim 19 wherein the fragments are derived from human immuno¬ globulins produced in tissue cultures, by human-mouse tri-hybridomas or by transformed B-cells.

21. The F(ab') or Fab' fragments of claim 18 wherein the fragments are produced by incubation of immunoglobulins in a conditioned medium acidified to pH 3.5.

22. A method for the production of F(ab') and Fab' fragments from an immunoglobulin containing hybridoma conditioned culture medium, said method comprising treat¬ ing said immunoglobulin containing medium with an immunoglobulin structural or conformation changing acid in an amount sufficient to lower the pH of the immunog¬ lobulin containing medium to a pH where substances pres- ent in the conditioned medium will convert the immunog¬ lobulin into the fragments.

23. A method in accordance with claim 22 wherein the acid is citric acid.

24. A method in accordance with claim 22 wherein after the conversion to F(ab') or Fab' fragments is substantially complete, the fragments are purified by diafiltration.