Large scale preparation of immunotoxins constructed with the Fab′ fragment of IgG1 murine monoclonal antibodies and chemically deglycosylated ricin A chain

Journal oflmmunologicalMethods, 112 (1988) 267-277 Elsevier

267

JIM04929

Large scale preparation of immunotoxins constructed with the Fab' fragment of IgG1 murine monoclonal antibodies and chemically deglycosylated ricin A chain * V. Ghetie, M.-A. Ghetie, J.W. Uhr and E.S. Vitetta Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75235, U.S.A. (Received 26 May 1988, accepted 3 June 1988)

In this report, we describe a method for the preparation of large amounts (grams) of immunotoxins (ITs) consisting of Fab' fragments of murine IgG1 monoclonal antibodies conjugated to chemically degiycosylated ricin A chain (dgA). The preparation of Fab' and dgA chain and the purification of the Fab'-dgA IT were accomplished by gel filtrations and affinity chromatography utilizing six Pharmacia Bioprocess columns (Sephadex G-25M, Sephacryl S-200HR and Blue Sepharose CL-4B) integrated into a semi-automatic chromatography system controlled by a Pharmacia C3-process controller. The final Fab'-dgA ITs were highly purified, potent, sterile and low in endotoxin concentration. Key words: Monoclonal antibody; Fab' fragment; Immunotoxin; Ricin toxin deglycosylated A chain; Large scale preparation of proteins

Introduction To construct an immunotoxin (IT) consisting of an antibody coupled to a toxin, there are several choices with respect to the molecular size and Correspondence to: E.S. Vitetta, Department of Microbiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, U.S.A. * Supported by NIH Grants no. CA-28149 and no. CA-41081, and a grant from the Welch Foundation (no. 1-0947). Abbreviations: A, ricin toxin A chain; B, ricin toxin B chain; dgA, deglycosylated ricin toxin A chain; DMF, dimethylformamide; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; F(ab')2 , Fab', pepsin fragments of IgG; Fab'dgA, immunotoxin containing Fab' and dgA; FACS, fluorescence-activated cell sorter; FDA, Food and Drug Administration; IT, irnmunotoxin; GMP, good manufacturing practice; LAL, Limulus amoeba lysate; PB, 0.05 M phosphate buffer, pH 7.0; PBE, 0.1 M phosphate buffer with 0.003 M Na2EDTA , pH 7.5; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

form of both components and the chemical linkage between them. An antibody can be used either as intact IgG or as fragments lacking the effector Fc region and carrying one (Fab') or two (F(ab')2) antibody combining sites. By eliminating the Fc region from the IgG molecule, the F(ab')2/Fab' fragments lose their ability to interact with the Fc receptors on many cell types (Morgan and Weigle, 1987) and become more accessible to tissues outside the blood stream (Sutherland et al., 1987). Ricin, a plant toxin, can be used in its native (AB) form or in a smaller form containing only the toxic A chain. The carbohydrate moiety contained in the A chain can be removed by chemical procedures (Vitetta and Thorpe, 1985) or by using a recombinant form devoid of carbohydrate (O'Hare et al., 1988). By removing the lectin (B) chain from the toxin and the carbohydrate moiety from the A chain, the new molecule loses its ability to interact

0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

268 with both galactose-containing membrane glycoproteins on all cells and the receptors for mannose on cells of the reticuloendothelial system (RES). The linkage between intact IgG or its F(ab')2 fragment and holotoxin or its A chain is accomplished by heterobifunctional crosslinkers which introduce a disulfide bond between the two components (Cumber et al., 1985). The conjugation of Fab' fragment to a sulfhydryl-containing toxin or A chain can be accomplished through a disulfide bond derived using the available cysteine residues of both proteins (Raso and Griffin, 1980). A disulfide bond is critical between A chain and antibody moiety if the conjugate is to be cytotoxic. ITs constructed with a Fab' fragment and dgA chain have been extensively tested for cytotoxicity using human and murine tumor cells. The results have shown that, in vitro, these monovalent ITs are only 2-10-fold less toxic than ITs containing IgG and that some Fab'-dgAs are as toxic to target cells as intact ricin (Ghetie et al., 1988; Shen et al., 1988). When Fab'-anti-6 ITs were administered in vivo to tumor (BCL1) bearing mice, they were more effective than IgG-anti-& dgA in localizing to tumor masses (Fulton et al., 1988a) and as effective at killing tumor cells (Fulton et al., 1988b). Moreover, the disulfide bond between Fab' and dgA was more stable than that of the PDP linker between the IgG and dgA (Fulton et al., 1988a). Therefore, Fab'-dgA ITs directed against B lymphoma cells are a candidate for therapy of B cell tumors in humans (Fulton et al., 1988b; Ghetie et al., 1988). To this end, we have constructed a scale-up good manufacturing practice (GMP) laboratory (Simmons, 1987) which can generate gram quantities of highly purified, endotoxin-low, sterile Fab-dgA in the amounts necessary for the in vivo therapy of B lymphoma in humans. In this paper, a standardized procedure for the large scale preparation of Fab'-antiCD22-dgA ITs is presented along with a short description of the physico-chemical and biological properties of the ITs. Materials and methods

Antibodies Hybridoma cells secreting the mouse IgG1 antiCD19 (HD37) and anti-CD22 (HD6) were gifts

from Drs. G. Moldenhauer and B. D6rken (Heidelberg, F.R.G.). Hybridoma cells secreting mouse IgG1 anti-CD22 (RFB-4) were obtained from Dr. G. Janossy (London, U.K.). Endotoxinlow (2 E U / m l / 1 0 mg) purified IgG1 antibodies from these hybridoma cells were produced by Damon Biotech., Needham, MA, and contained less than 5% impurities as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein was dissolved in 0.01 M phosphate buffered saline, pH 7.2 and stored at -70°C.

dgA The dgA chain of ricin was purchased from Inland Laboratories, Austin, TX, and was prepared and characterized as described by Fulton et al. (1986). The effectiveness of the deglycosylation procedure was determined by demonstrating that 10% or less of the dgA chain (as compared to more than 90% of native A chain) could bind to concanavalin A-Sepharose 4B (Sigma, St. Louis, MO) in the presence of 0.1 M galactose. The protein was dissolved in phosphate buffered saline, pH 7.2, containing 50% glycerol and stored at -20°C.

Preparation of ITs All procedures were performed in a GMP laboratory with sterile, endotoxin-free distilled water (obtained by reverse osmosis), buffers and equipment using a chromatographic system depicted in Fig. 1. The six steps of the procedure (see Fig. 2) were performed in 3 days.

Preparation of Fab' fragments 4 g of the antibody is brought to 2.5 mg/ml in 0.1 M citrate buffer pH 3.7 by dilution with 1 M citrate buffer (pH = 3.7) and distilled water. Pepsin dissolved in 0.1 M citrate buffer pH 3.7 is added to the antibody solution (20 mg pepsin/g IgG1) and the digestion is performed at 37 °C for 2-8 h with occasional stirring (2 h for HD37, 6 h for HD6, and 8 h for RFB-4). The pH of the digest is then brought to approximately 8.0 with 1 M sodium hydroxide. The digest is applied to a Sephacryl S-200HR column equilibrated in 0.1 M phosphate buffer with 0.003 M Na2EDTA (PBE) and the F(ab')2 peak is collected (Fig. 3B) and

269

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c o n c e n t r a t e d to 5 - 1 0 m g / m l b y C H 2 spiral c a r t r i d g e c o n c e n t r a t o r Y30 ( A m i c o n , Denvers, M A ) at 4 ° C. T h e F a b ' c o n c e n t r a t e is b r o u g h t to r o o m t e m p e r a t u r e a n d d i t h i o t h r e i t o l ( D T T ) is a d d e d to a final c o n c e n t r a t i o n of 5 m M . T h e r e d u c tion with D T T p r o c e e d s for I h at 2 0 - 2 5 o C in the dark. T h e solution of r e d u c e d F ( a b ' ) 2 is cooled on ice a n d c h r o m a t o g r a p h e d o n a S e p h a d e x G - 2 5 M c o l u m n . T h e F a b ' fraction is c o n c e n t r a t e d to 5 - 1 0 mg/ml a n d 5 , 5 ' - d i t h i o b i s ( 2 - n i t r o b e n z o i c acid) ( D T N B ) dissolved in d i m e t h y l f o r m a m i d e ( D M F ) is a d d e d to 2 m M . T h e m i x t u r e is i n c u b a t e d for 1

h at 2 0 - 2 5 ° C a n d then c o o l e d a n d c h r o m a t o g r a p h e d on a S e p h a d e x G - 2 5 M c o l u m n equil i b r a t e d with PBE. T h e p e a k d e v o i d of a n y free D T N B is collected (Fig. 3A) a n d c o n c e n t r a t e d to a p p r o x i m a t e l y 4 m g / m l . T h e m i x t u r e is m a i n t a i n e d on ice for n o longer t h a n 2 h before rea c t i n g it with r e d u c e d A chain. T h e yield of the F a b ' p r e p a r a t i o n is 2 g.

Preparation of reduced dgA chain T h e p H of d g A c h a i n s o l u t i o n (5 m g / m l , app r o x i m a t e l y 2 g) is b r o u g h t to p H 7.5 with 1 M

270

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N a O H and D T T is added to a final concentration of 5 raM. The solution is incubated in the dark for 1 h at 2 0 - 2 5 ° C and then cooled and chromatographed on Sephadex G-25M equilibrated with PBE to remove free DTT. Finally, the solution is concentrated to approximately 4 m g / m l in a CH2 spiral cartridge concentrator Y10.

Conjugation of Fab'-TNB with dgA-SH The p H of both solutions should be 7.5 (PBE) (adjusted with 1 M sodium hydroxide). The reduced dgA-SH chain (2 g) is added to the Fab'T N B derivative (2 g) and the solution is mixed by gently stirring in a horizontal shaker for 2 h. The solution becomes yellow as the F a b ' - T N B conjugates to the dgA-SH (elimination of TNB). The optical density of the mixture at 412 nm should be 0.5. The mixture is dialyzed for 16-20 h at 4 ° C against 0.05 M phosphate buffer (PB) p H 7.0 in boiled dialysis tubings.

Purification of Fab'-dgA The crude conjugate (approximately 4 g in no more than 2 liters) is applied to the Blue-Sepharose CL-6B column (Knowles and Thorpe, 1987)

271

equilibrated with PB. The fraction eluted with the buffer is discarded since it contains unconjugated Fab' fragment and some inactivated dgA chain. The fraction eluted with 1 M sodium chloride (Fig. 3C) is concentrated and chromatographed on a Sephacryl S-200HR column equilibrated with 0.145 M sodium chloride. The first peak (Fig. 3D) contains the conjugate and the second peak contains dgA. The Fab'-dgA conjugate is concentrated to 2-3 mg/ml and is sterilized through a 0.22/~m disposable sterile filter (Coming Glass Works, Corning, NY). Samples are aliquoted into endotoxin-free vials (Wheaton serum bottle, Southland Cryogenics, Carrollton, TX) at 10-50 mg/vial and sealed in a laminar flow hood. The vials are immediately snap-frozen at - 7 0 °C and can be stored at this temperature for at least 1 year without any change in activity.

Analysis of Fab'-dgA immunotoxins A cell-free rabbit reticulocyte assay, a cell killing assay, indirect immunofluorescence (FACS) and LDs0 determinations (in mice) were described in previous studies (Press et al., 1986; Ghetie et al., 1988). SDS-PAGE was carried out using the Pharmacia Phast system with a 8-25% gel gradient. The gels were stained with either 0.1% Phast gel blue R or with silver nitrate as indicated by the manufacturer. The Limulus amoeba lysate (LAL) assay was used to detect endotoxin according to the techniques recommended by the manufacturer (Associates of Cape Code, Woodhole, MA). The results were expressed in EU/ml, one endotoxin unit being described as the potency of 0.2 ng endotoxin. Clearance of Fab'-dgA from the blood of mice was carried out as described (Fulton et al., 1988a).

TABLE I C H A R A C T E R I S T I C S OF T H E C O L U M N S U S E D F O R L A R G E SCALE P R E P A R A T I O N O F Fab'-dgA I M M U N O T O X I N S Parameter

Gel Bed volume (1) Void volume (1) M a x i m u m loading volume (1) HETP b M a x i m u m flow rate ( l / h ) Pressure (MPa) Equilibrated with: Preparation step e

Used for r

Pharmacia Bioprocess column, type (cm) 252/60

252/30

116/30

Sephacryl S-200HR 30 9.0

Sephadex G-25M 15 4.5

Blue Sepharose CL-6B 3.5 0.9

0.6 0.05 5 0.06 PBE c Saline d 2-3 7-8 F(ab') 2 separation Free A chain removal from IT

0.3 0.14 10 0.15 PBE 3-4 4-5 4'-5' D T T removal from Fab' D T N B removal from Fab' D T T removal from A chain

2.0 a 0.05 2 0.03 PB e Eluted with 1 M NaC1 6-7

Free Fab' removal from IT

a The binding capacity of the column is 3 g A chain. Therefore, the loading volume was usually 2 liters containing approximately 2 g of free and IT bound A chain. b Height equivalent to a theoretical plate was calculated according to the formula: H E T P = L / N (cm); N = 16 ( V e / W ) 2 where .L(cm) = colunm length; W (1) = peak width; Ve(1) = elution volume of NaCI; N = n u m b e r of theoretical plates. c 0.1 M phosphate buffer with 0.003 M N a 2 E D T A , p H = 7.5 (buffer 1 in Fig. 1). d 0.145 M NaC1 (buffer 3 in Fig. 1). e 0.05 M phosphate buffer p H = 7.0 (buffer 2 in Fig. 1). f See Fig. 2.

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Columns The characteristics of the Pharmacia Bioprocess columns used for the preparation of IT are presented in Table I. In some instances, for the separation of IT from free A chain, a 10/90 cm Sephacryl S-200SF column was used when the amount of Fab'-dgA conjugate was under 1 g. For the estimation of the percentage of F(ab')2 released after digestion of IgG (Fig. 4) and for the percentage of Fd' + L chain released after reduction of Fab' fragment (Fig. 5), chromatography on small columns of Sephacryl S-200HR and SDSP A G E analysis, respectively, was used. v

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Results

The procedure The large scale preparation of a Fab'-dgA conjugate follows the procedure already described for the preparation of small amounts (rag) of antiCD22 (HD6 and RFB-4) murine monoclonal IgG1 antibodies and anti-CD19 (HD37) murine monoclonal IgGl antibody (Ghetie et al., 1988; Shen et al., 1988). The flow sheet for large scale preparation of Fab'-dgA is presented in Fig. 2 and shows that the Fab' fragment is obtained by digestion of IgG1 with pepsin followed by reduction with D T T and protection of SH group by D T N B while dgA chain undergoes reduction with DTT. During preparation of both Fab' and dgA chain, the excess D T T or D T N B is removed by gel filtration on Sephadex G-25M columns followed by concentration of the protein solution to 5-10 mg/ml. After conjugation of Fab'-TNB and dgA-SH, the free dgA chain and Fab'-TNB are removed by a twostep purification procedure using affinity chromatography on Blue Sepharose CL-6B and gel filtration on Sephacryl S-200HR. There are three critical steps in the preparation of Fab'-dgAs which should be carefully controlled for each monoclonal antibody used and for the resulting immunotoxin. The first critical step in the procedure is the pepsin digestion (steps 1 2 in Fig. 2) of mouse IgG1 monoclonal antibody, a process which requires different periods of time for each monoclonal antibody, even of the same is•type. To establish the optical conditions for digestion (e.g., the maximum yield of F(ab2), several parameters should be established including pH, time of incubation, protein concentration and enzyme/substrate ratio. In Fig. 4, an example is given illustrating how we established the optimal conditions for the digestion of monoclonal IgG1 antibodies (pH = 3.7, concentration = 2.5 mg/ml, time = 2-8 h, and enzyme/substrate = 2/100). The second critical step is the reduction of F(ab')2 fragments (steps 3 - 4 in Fig. 2); reduction of the molecule to Fd' and L chain components is to be avoided. An appropriate concentration of DTI" should be used to minimize the amounts of free L chain and Fd' fragment. The example given in Fig. 5 shows that a final concentration of 5 mM

273 TABLE II THE EFFECT OF pH AND MOLARITY ON THE BINDING OF Fab'-dgA TO BLUE SEPHAROSE CL-6B Buffer Phosphate Phosphate Phosphate Phosphate + Na 2EDTA Phosphate + Na2 EDTA Phosphate + Na 2EDTA

Molarity (M)

pH

0.05 0.05 0.1 0.05 0.1 0.1

6.5 7.0 7.0 7.5 7.5 8.0

% Bound fab'-dgA

a

> 95 > 95 78 66 27 b <5

a The capacity of Blue Sepharose CL-6B at 0.05 M phosphate buffer pH 7.0 was 1 mg A chain/ml packed gel. b Some batches of Blue Sepharose CL-6B were able to bind > 95% of Fab'-dgA.

D T T is appropriate for generating the optimal amount of Fab' while keeping the splitting of the moleeule into F d ' and L chain components at less than 10%. The number of SH groups released during the reduction with 5 m M D T T never exceeded 2, an average of 1.4 + 0.2 was recorded in 12 experiments performed with all three monoclonal antibodies and the IgG1 myeloma protein MOPC-21 control (Ghetie et al., 1988). The third critical step is the interaction of Fab'-dgA with Blue Sepharose CL-4B (steps 6 - 7 in Fig. 2). As indicated by the results presented in Table II, there should be strict control of the p H and molarity of the mixture. Since the reaction between F a b ' - T N B and dgA-SH takes place at p H 7.5 in 0.1 M PBE, the molarity and the p H of this mixture was adjusted to 0.05 M and p H 7.0 after completion of the reaction (2 h) by overnight dialysis at 4 ° C against an appropriate phosphate buffer (0.05 M PB, p H = 7.0).

The structure of the chromatographic system The preparation of F a b ' - T N B and dgA-SH as well as the purification of the Fab'-dgA conjugate involves the use of six Pharmacia Bioprocess columns as indicated in Table I. The integration of these columns in the chro~natographic system was achieved in a semi-automatic circuit containing pumps, air sensors, flowstoppers, air traps, flowmeters, UV monitors (and recorders), and five automatic solenoid valves as

depicted in Fig. 1 (continuous fine). Each column was connected to this standard system when the respective separation step was reached and the chromatographic process was continuously controlled by a Pharmacia C3 process controller which could read and respond to the following events: buffer delivery to the automatic three-way valves, the o n / o f f speed of the pump, the presence of air bubbles in the system (air sensor), the variation of the flow rate (flowmeter, flow-stopper), the increase vs. decrease of the protein concentration (UV monitor), and the collection of protein-containing fractions (Fig. 1, dotted lines).

The function of the chromatographic system The buffers are maintained at room temperature in nonsterile, endotoxin-free cylindrical polyethylene tanks (114 L) (Fisher Sci., Pittsburg, PA). F r o m the barrels, the buffers are p u m p e d with a Watson-Marlow 501U p u m p through a Versaflow capsule filter (0.45 mM) (Gelman Sci., Ann Arbor, MI) into 20 liter sterile glass water bottles (American Sci. Products, M c G a w Park, IL) maintained in cold boxes at 4 ° C . From the glass reservoirs, the buffers are pumped by another p u m p to the system containing the Pharmacia Bioprocess column and all the devices necessary for the functioning of this column (see Fig. 1, continuous fine) maintained together at 4 ° C in cold boxes. The colunms are loaded with a protein sample by activating valves V1 and V4 and the separation process is automatically continued until the protein fraction is eluted from the column. The protein fraction is directly collected (by C3 process controller-activated valve V5) into a CH2 concentrator provided with a spiral cartridge filter (Fig. 1). After concentration, the sample is introduced into another type of column using the same chromatographic system and the process is repeated with all six columns (Table I).

Chromatographic resolution The separation of proteins from excess D T T or D T N B was achieved with very good efficiency; no traces of free D T T or D T N B were detected in the gel-filtered solution. The difference between the elution volume of proteins and D T T or D T N B was over 5 liters (on 2 5 / 3 0 cm Sephadex G-25M columns) (Fig. 3A). This is important since traces

274

1

2

A

I~

C

D

E

Fig. 6. SDS-PAGE of Fab'-dgA and its components. Lane A = standards (l = 20 kDa: 2 - 30 kDa; 3 = 46 kDa; 4 = 68 kDa; and, 5 - 94 kDa). Lane B = Fab'-TNB. Lane C = dgA chain. Lane D = immunotoxin. Lane E = immunotoxin reduced with 5% 2-mercaptoethanol (contains dgA chain, L chain and F d ' fragment, the last two are not separated from each other).

of DTNB in Fab'-TNB and DTT in the reduced A chain may result in low yields. The separation of F(ab')2 fragments from undigested IgG as well as from pepsin and the low molecular fragments (resulting from the splitting of the Fc region) is achieved by gel filtration on Sephacryl S-200HR column (Fig. 3B). Since the separation of IgG (MW 150000) from F(ab')2 (MW 100 000) is not complete (for a 60 cm length column), we accomplish the digestion such that less than 5% of intact IgG remains in the digest (Fig. 4). Moreover, the proteins eluted in the first fractions of the ascending slope of the F(ab')2 are discarded. We also discarded the proteins eluted in the last fractions of the descending slope of the F(ab')2 to ensure that no pepsin is present in the

F(ab') 2 fraction. This is controlled for each batch with an anti-pepsin antibody by a double diffusion gel assay. However, a protein fraction with MW 60 000 is always present as a 5-10% impurity in the F(ab')2 preparation of all antibodies (HD6, RFB-4 and HD37). The resolution of the affinity chromatography procedure on Blue Sepharose CL-6B is excellent and results in complete elimination of almost all protein not containing A chain (e.g., free Fab') (Fig. 3C). The final chromatography step on Sephacryl S-200HR results in the separation of Fab'-dgA from A chain (monomeric and dimeric) and some high molecular weight conjugates, respectively. However, the separation is not complete as illustrated in Fig. 3D and, therefore, both high molecular weight con-

TABLE III P R O T E I N LOSSES D U R I N G Fab'-dgA P R E P A R A T I O N D U E TO C O N C E N T R A T I O N O F P R O T E I N S O LU TIO N S Concentrator

Filtron Amicon Amicon Amicon Amicon

8050 8200 CH2 DC10L

Filter type

Omega 10-30 PM 10-30 YM 10-30 Spiral Crt Spiral Crt

Capacity (ml)

Percentage of antibody lost after concentration a Steps 2 - 3

Steps 4 - 5

Steps 6 - 8

Total loss

10 50 200 2 000 20000

25 30 24 18 ND c

10 20 16 15 ND

19 28 21 17 ND

54 78 61 50 75 b

a For steps, see Fig. 2. b Six successive concentrations of human IgG from 1 m g / m l to 10 m g / m l . c Not determined.

275

jugates (containing more than one molecule of A chain per Fab') and free A chain are present in the final preparation at a level of 5-15% (Fig. 6). The final gel filtration on Sephacryl S-200HR is performed in 0.145 M sodium chloride to allow, after concentration and sterilization, the direct intravenous use of ITs for in vivo therapy.

Yields Since the procedure is based almost entirely on gel filtration, the major sources of protein losses are the concentration steps. A significant amount of protein is lost due to either the adsorption of proteins on the filter (hydrophobic dgA chain and Fab'-dgA) or to the dead volume of the spiral cartridge concentrators. The results presented in Table III were obtained by using concentrators with different process volumes for the preparation of ITs ranging from 5 to 1000 mg. For large scale operation, the total loss of protein is as high as 50%. This low recovery of protein after six successive chromatography and concentration procedures is the major cause for the low yields of ITs preparations. These yields were never higher than 25% of the amount of IgG1 used for pepsin digestion (Table IV). Process hygiene in I T preparation The safety of the final product depends not only on the non-specific toxicity of the IT but also on the removal of bacteria and their by-products TABLE IV YIELDS D U R I N G LARGE SCALE PREPARATION OF Fab'-dgA IMMUNOTOXINS a Step of preparation b Pepsin digestion of IgG1 (steps 2-3) Preparation of Fab'-TNB (steps 4-5) Purification of immunotoxin (steps 6-8)

Actual yield (%)

Theoretical yield

50

66

42

60 c

25

48 d

" Expressed as percentage of the initial amount of IgG1 antibody used for pepsin digestion. b See Fig. 2. c Assuming that 10% is lost by reduction. d Assuming that the yield of Fab'-dgA formation should be 80% (Raso and Griffin, 1980).

TIME OF INCUBATION (h) ( 0 5 M NaOH at 4"C) t2 (.24 i

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t0

NaOH Molarity (M) (4Bh at 4*C) Fig. 7. Inactivation of endotoxin by sodium hydroxide treatment. • • , NaOH molarity (M); o o, time of incubation.

(e.g., endotoxin). This was accomplished by cleaning the system before use with 0.5 M sodium hydroxide (Curling et al., 1982; Sofer, 1984). All tubing, valves, and columns washed in N a O H for 48 h at 4 ° C were low in endotoxin as shown in Fig. 7. The sodium hydroxide was washed out from the system at the maximum flow rate permitted using the appropriate buffer. The conditions were applied for all Sephadex G-25M and Sephacryl S-200HR columns except for Blue Sepharose CL-6B which was cleaned with 0.2 M sodium hydroxide for 48 h at 4°C. All immunotoxins obtained by G M P scale-up procedure were sterile but contained varying amounts of endotoxin (1-15 E U / m g IT) irrespective of the fact that the chromatographic system (concentrators included) was endotoxin free prior to use. These results suggest that in such cases contamination may have occurred during the manipulation of the protein solutions outside the chromatographic system. The highest concentration of endotoxin recorded in all the experiments performed to date was 15 E U / m g which is approximately two-fold higher than the limit set by the FDA (MBller-Cogan, 1987; Munson, 1987) if a 50 mg dose of IT is given to a 70 kg patient (tolerance limit (FDA) 5 E U / k g ; 15 EU x 50 = 750 EU; 750 E U / 7 0 kg = 10.7 EU/kg).

276 TABLE V

IT). Less than 1% of the ITs were b o u n d to thiopropyl-agarose (Table V).

ANALYSIS OF Fab'-ANTI-CD22-dgA Assay

Monoclonal antibody used to prepare Fab'-dgA

Sterility LAL assay (EU/ml) SDS-PAGE (% of major 80 kDa band) Binding to thiopropylSepharose 6B Reticulocyte assay (M) Daudi-killing assay (M) b Inhibition of antibody activity by binding Fab' to dgA (%) LDs0 (rag/25 g mouse) e Clearance (h) f a

HD6

RFB-4

Negative 1.0

Negative 5.0

85

90

No 2.9×10 11 6.7×10 -11

No

40 c 1.5 2.0

60 d 1.5 1.5

1.5 ×10--11

1.2×10 -11

For free dgA chain, the values were between 10-n-10 12 M. b For free dgA chain, the values were 10 7 M. The Fab'-TNB was not toxic for cells. c Concentration giving 50% fluorescent cells: Fab'= 0.6 /~g/ml=1.2×10 -8 M; Fab'-dgA=l.6 /~g/ml=2×10 -8 M; Fab'/Fab'-dgA = 1.2/2.0 = 0.6 × 100 = 60%; 100%- 60% = 40%. a Concentration giving 50% fluorescent cells: Fab'= 0.6 /~g/ml =1.2×10 -8 M; Fab'-dgA = 2.4 /Lg/ml = 3x10 8 M; Fab'/Fab'-dgA = 1.2/3.0 = 0.4 × 100 = 40%; 100%- 40% = 60%. The proportion of dgA chain in conjugate is 40%; the LDs0 for dgA chain was approximately 0.5 mg/animal. f In mice. Comparable half-lives were recorded for dgA and Fab'-TNB.

The properties of ITs The routine checking of the I T preparations obtained by the large scale procedures utilizes the assays presented in Table V. The inactivation of a n t i b o d y was determined by an indirect technique which measured the binding of the F a b ' vs. F a b ' - d g A conjugate to D a u d i cells (Ghetie et al., 1988). The results show that the percentage of a n t i b o d y inactivated after conjugation is approximately 50%. The presence of SH group in the ITs was determined using D T N B (Ellman, 1959) and by affinity c h r o m a t o g r a p h y on thiopropyl-agarose (Sigma, St. Louis, M O ) (Hilson, 1981). In b o t h assays free SH groups were not detected in the I T (under 0.1 SH g r o u p / m o l e c u l e

Discussion The preparation of ITs using the F a b ' fragment of m o n o c l o n a l a n t i b o d y and the dgA chain of ricin can be scaled up to obtain large a m o u n t s of ITs (grams) without imparing the purity of the final p r o d u c t or decreasing the yield of the preparation as c o m p a r e d to essentially similar procedures (Ghetie et al., 1988; Shen et al., 1988) utilized for the preparation of ITs in small amounts (mg) for experimental purposes. The c h r o m a t o g r a p h i c system used for the preparation of ITs involved six steps for the F a b ' - T N P and d g A - S H preparations, their conjugation and final purification. Each step is controlled by a process controller which is p r o g r a m m e d to ensure the continuous flow of the separation procedure. However, the process was interrupted twice during the separation: after the F(ab')2 purification and after the conjugation. The F a b - d g A can be prepared in 3 days. During the first day, the digestion of I g G and the purification of F ( a b ' ) 2 takes place. The second day is used for the preparation of F a b ' - T N B and d g A - S H and their conjugation. After an overnight dialysis, the last d a y is used for the purification of the immunotoxin. The sterilized preparations are maintained at - 7 0 ° C since, at this temperature, the shelf life of the conjugates is longest (at least 1 year). The preparations obtained by the procedure described are sterile and endotoxin free, two quality parameters strictly required for their use in humans. The biological activity of the I T was measured by its ability to bind to cell surfaces (FACS), to inhibit protein synthesis (after reduction) in a cell-free assay and to kill t u m o r cells bearing the relevant antigen in vitro. The values obtained d e m o n s t r a t e that F a b ' - d g A is a very potent IT when the m o n o c l o n a l antibodies are directed against epitopes on CD22. The ability of these F a b ' - a n t i - C D 2 2 - d g A to inhibit protein synthesis and to kill target cells is c o m p a r a b l e to that of intact ricin (Ghetie et al., 1988; Shen et al., 1988). Therefore, these ITs are candidates for use in h u m a n s with refractory B cell l y m p h o m a and leukemia (Ghetie et al., 1988; Shen et al., 1988).

277

Acknowledgements W e t h a n k Ms. R. Nisi, Ms. B. S m i t h , M r . S. C h i n n , M r . T. T u c k e r , a n d Dr. D . R i c h a r d s o n for e x p e r t t e c h n i c a l a s s i s t a n c e a n d Ms. C i n d y B a s e l s k i for s e c r e t a r i a l assistance. W e e s p e c i a l l y t h a n k M r . P e t e r G a n i o n for his h e l p f u l s u g g e s t i o n s o n t h e l a r g e scale p r e p a r a t i o n o f p r o t e i n s .

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