Immunoaffinity chromatography of clinical products

TIBTECH - APRIL 1987 [Vol. 5]

DDII DB Im ll li Immunoaffinity chromatography of clinical products G. W. Jack and H. E. Wade The high specificity of antibodies for antigens has long attracted the interest of scientists in the field of protein purification. The rapidly increasing availability of pure monoclonal antibodies has stimulated their use in the immunoaffinity chromatography of therapeutic proteins and the interests of licensing authorities. Scientists interested in the purification of natural substances have been quick to scrutinize and exploit every advance in immunology to take advantage of the high specificity of antibody for antigen. Initially bacterial or viral antigens were precipitated in solution using natural (polyclonal) antibodies, but this approach suffered the practical disadvantages of requiring a specific ratio of antibody:antigen to avoid solubilization by excess antigen 1'2 and demanded rather severe conditions for dissociation imposed by the very high affinities expressed by most preparations of polyclonal antibody. The immobilization of one or other of the components eased this problem and Campbell and his colleagues3 very successfully purified antibodies on cellulose-bound antigens. The prospects for the reverse application (antigen purified on immobilized antibody) were entertained in several reports towards the end of the 1950s but the greatest interest at that time remained the fractionation of antibodies and many matrices for antigen attachment were introduced including cation-exchange resins,

polystyrene and polyacrylamide derivatives4. Even after immunization with a very pure antigen, the antibodies produced by an animal comprises a heterogeneous population of immunoglobulins with a variety of affinities for the antigen and a host of other antibodies already circulating in the animal. With the development of monoclonal antibodies (MAbs) by Milstein 5, there was less demand for

fractionating antibodies because the higher degree of specificity sought could be achieved with antibodies from hybridomas. The general availability of monoclonal antibodies has had an enormous impact on practically every field of biological science and not least on the immunoaffinity chromatography (IAC) of proteins for therapeutic uses (Fig. 1). The absolute specificity of MAbs for a single epitope (the smallest immunologically recognized sub-molecular group on an antigen) enabled researchers to select from a panel of MAbs the one most suited to antigen purification from those produced by other hybridoma clones which, perhaps through a higher affinity for antigen had a more promising future in immunoassay. Affinity

MAbs have affinities (see Glossary) for their antigens ranging from 107 to 1011 I mole-1 which represent high binding efficiencies for antigen and an ability to concentrate antigens from very dilute solutions. Staehelin et al.,6 for example reported the use of a 17 ml column of immobilized MAb for the purification of the 30 mg of recombinant human leukocyte interferon present

- Fig.1 0 o

~']l'~~g

~ E~

Washing~L (~~'~O'(~o EluUon

G G. W. Jack and H.E. Wade are at the PHLS Therapeutic Products Laboratory, Centre for Applied Microbiology and Research, Porton, Salisbury, UK.

(~/~

Key

Matrix AntibodyAntigen %

Immunoaffinity chromatography. © 1987, Elsevier

Publications,Cambridge

©

0166 - 94301871502.00

TIBTECH - APRIL 1987 [Vol. 5]

- - Table I

Proteins of potential therapeutic value which have been purified by immunoaffinity chromatography on monoclonal antibody columns

in 1 kg of frozen bacterial cell paste obtaining a purification in excess of one thousand-fold. One of the major problems with protein purification, the dissociation of the antibody-antigen complex without damaging the components, can be eased by choosing an MAb of relatively low affinity: of the order 10 8 1 mole -1. For the preparation of therapeutic proteins however a different although equally demanding challenge is that of satisfying Medicines Regulatory Authorities who are charged with the clinical safety of products manufactured by this route. The range of licenced products in current clinical use which have been ~ purified on immobilised MAb columns is hard to assess because of the natural reluctance of pharmaceutical companies to divulge their methods. Hoffmann La Roche, however, has produced commercial amounts of recombinant human leukocyte interferon 2A 6 by a combination of MAbbased IAC and metal chelate chromatography and both urokinase 7 and Factor VIII/van Willebrand 8 complex are products of this technique. If to these are added products currently undergoing clinical trials and those potential clinical products whose

Protein

Potential use

Ref.

Human interferon Glycoprotein D of herpes simplex (HSVl and HSV2)

Immuno-regulation Vaccine

9 10

Recombinant hepatitis B antigen Erythropoietin Human renin

Vaccine Erythropoiesis stimulation Coronary insufficiency Treatment of cancer Dissolution of blood cells Prothrombin cleavage Tissue stimulation

11 12 13 14 15 16 17

Interleukin-2

Tissue plasminogen activator Human factor X Somatomedin C

purification by this route has been reported, a substantial list can be compiled (Table 1). Projecting into the future, a significant expansion may be expected with the purification of antigens from viral coats and microbial envelopes for new vaccines. Some insight into the high degree of selectivity which may be obtained with MAbs comes from the work of this laboratory. We have used immobilized MAb columns to resolve human pituitary glycoproteins: a mixture of proteins sharing a high degree of homology. The hormones, Thyroid Stimulating Hormone (TSH), Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) are dimeric proteins sharing a common subunit with the uncommon subunits showing a high degree of sequence homology 18. Nevertheless, suitable MAbs were found capable of producing pure preparations of each hormone substantially free of contamination from the other hormones 19.

Preparation and operation of MAb columns Two commercially available support matrices, both based upon agarose, are most commonly used in IAC. CNBr-activated Sepharose (Pharmacia) is available in both a crosslinked and noncross-linked form and can be used to generate an isourea linkage under mildly alkaline conditions with alkylamino groups on the protein surface. AffiGels (BioRAD) are N-hydroxysuccinimide esters of a derivatized cross-linked agarose gel: on coupling at pH values near neutrality Nhydroxy-succinimide is displaced and a stable amide bond is formed with free alkyl or aryl amino groups. With both products the linkage formed between support and protein is stable over a wide pH range, but in

the case of the isourea bond it is more stable at acid pH. More recently Pharmacia has introduced Tresyl-activated Sepharose for immobilizing proteins through either free amino or sulphydryl groups while LKB has marketed Trisacryl, a synthetic poly-Nacryloyl-2-amino-2-hydroxymethyl1,3 propanediol which is activated by a range of chemistries. The Fractogel TSK series, available from Merck, is a hydrophilic vinyl polymer which again may be derivatized in a variety of ways. With all these matrices, one of the major problems is leaching of MAb during product elution. However, since this is performed mainly at an acid pH or at neutral pH in the presence of chaotropes (see Glossary), contamination of the product from this source is minimized.

Antibody-antigen binding The conditions under which IAC is performed can have a profound bearing on the quality of the product 2°. The nature of the buffer used during the binding of antigen to immobilized antibody can influence the quality of the product: because of its amphoteric nature, an immobilized protein acts as mixed ionexchanger; it also presents a hydrophobic site of attachment accentuated in the case of Affi-Gel matrices by the presence of an aliphatic spacer arm. Non-specific binding to IAC columns may be minimized by performing the antigen-antibody binding step in buffers of high ionic strength containing a non-ionic detergent 21 providing the presence of the detergent does not compromize the use of the product as a therapeutic product.

Dissociation The dissociation of the antigenantibody complex is arguably the

TIBTECH -APRIL 1987 [Vol. 5]

most critical step in IAC. It is most commonly performed by the use of low pH (pH 2-4) occasionally in the presence of denaturing agents such as urea or guanidinium. Alternatively concentrations of chaotropic agent such as iodide or thiocyanate of up to 3 M may be used at neutral pH. The choice of conditions is dictated by the stability of the antigen and the ease with which the antigen-antibody complex can be disrupted. With sensitive proteins a compromise may have to be made with respect to the affinity of MAb chosen for the purification; a MAb of low affinity will permit mild conditions of elution but the resulting MAb-matrix may have relatively low antigen binding capacity. However as in all systems of protein purification, problems occur and it w o u l d be unrealistic to expect recoveries in excess of 90%. N e w elution solutions

A number of novel approaches to the elution problem have been described recently. In one, MAbs were selected with a positive temperature-dependent affinity 22 a consequence of which was that, after generating the antigen-antibody complex at room temperature, the antibody could be eluted by lowering the temperature to 4°C. Another approach is applicable to proteins with a specific metal ion 23 or cofactor requirement. Both Prothrombin and Factor IX have been purified on MAbs which recognize only the Ca2+-stabilized forms of these molecules. After forming the antigenantibody complex in the presence of Ca 2+, the antigen could be isolated by elution with EDTA. This very elegant solution is restricted to proteins that undergo a considerable change in tertiary structure upon removal of the metal ion or co-factor (abolishing epitopes on the apoprotein) and to proteins that are stable as both apoprotein and holoprotein. This approach however offers the possibility of product elution from IAC columns of highly sensitive proteins under conditions that are unlikely to damage antigen or MAb. Another strategy, applicable to recombinant proteins is to fuse an enzymatically-cleavable peptide to

the N-terminus of the protein and perform the purification of the fusion product on an MAb column directed at the peptide 24. Once purified, the desired protein is obtained by enzymatic cleavage. In such a system, only one MAb need be selected to effect the purification of a wide variety of recombinant proteins. Irrespective of the precise nature of the elution process, there i s an advantage in eluting the antigen from the IAC column in the reverse direction to that used for loading. Since columns are normally loaded well below the equilibrium capacity of the matrix, eJuting in the same direction can result in the antigen reacting with unoccupied sites as it moves d o w n the column, resulting in a broadening of the elution profile. In contrast, with reverseflow elution, much narrower peaks of eluting antigens are obtained. The method also has the advantage of cleaning the surface of the gel bed of any protein that precipitates during loading. C o l u m n lifetime

By their biological nature, IAC columns are highly susceptible to proteolytic and microbial degradation. In the course of manufacturing therapeutic products, the columns are run using sterile reagents but in our experience it has been found advantageous to run even experimental chromatographic separations in a clean-room environment as this greatly extends the working life of the MAb-matrix (absorbent). Operating under these conditions, the longevity of the absorbent can be very impressive. Reports regularly appear of their use for more than a year and in excess of a hundred cycles of operation and the large amounts of product obtained comfortably offset the high initial cost. Should it be felt necessary to include a preservative in buffers used for column storage, the choice should be one compatible with pharmaceutical manufacture. Bacteriostats such as chlorhexidine (hibitane) and protease inhibitors such as aprotinin are likely to be acceptable to the Regulatory Authorities. With commercially available mat-

rices, reproducible synthesis of MAb-matrices is readily achieved and should a batch of absorbent become fouled, a replacement batch w o u l d be expected to behave identically in the process. This is the more important for therapeutic substances of ill-defined molecular structure where reproducibility of the process assumes a greater importance in a presentation to the Regulatory Authorities. For large-scale operations the use of cross-linked matrices is advisable since at high linear flow rates, they compress much less readily and maintain better flow characteristics than their noncrosslinked counterparts. A u tom a tion

IAC readily lends itself to automation. Both matrix and MAb are relatively expensive, hence the repeated use of a relatively small IAC column is preferable to a large one used less frequently both with respect to the initial cost of the column and its possible deterioration during storage. This choice is encouraged by the numbers of programmable systems that are available commercially (such as the Pharmacia C3) and described in the literature 19'25. All these systems activate pumps and solenoid valves in a pre-determined sequence based either upon time or liquid flow and are inexpensive. About £500 will provide a programmable system 19 capable of controlling eight pieces of apparatus and providing sixteen individuallytimed programmable steps. Minicomputer-based systems are now being developed 26 which interact with signals from equipment monitoring UV-absorbance, pH or conductivity; such systems can be used to optimize a separation during the development of the process and when applied to repetitive use for production can accommodate shifts in flow rate and peak widths. With the addition of suitable protective devices such as fluid sensors 27, automated systems can be run continuously with a considerable saving on production costs. Sequential purifica tion

With the high selectivity of MAbmatrices, the crude starting material

TIBTI=CH - APRIL 1987 [Vol. 5] - Fig. 2

Mixture of A,B,C and Contaminants

Column of MAb to A

for antigen purification p a s s e s through an IAC column unaffected except for the removal of a single protein species. Should the starting material contain more than one protein of interest these may be purified sequentially in a cascade system 19 (Fig. 2) where the unretarded fraction from one MAbmatrix is passed directly through a second column and then a third. This technique had been applied successfully in this laboratory to optimize the recovery of pituitary hormones from very valuable source material with the advantage that no processing is necessary between the various IAC columns 19.

The requirements of National Health Authorities Over the past five years National Health Authorities have responded to increasing clinical interest in monoclonal antibodies by drafting guidelines for their pharmaceutical manufacture. The guidelines are generally directed at their use as drugs for direct administration not at their use as reagents in the preparation of drugs and there are interesting differences in both scope and emphasis. The subject of the guidelines drafted by the EEC 28, for example, is confined to monoclonal antibodies from rodent sources. The USA guidelines issued by the FDA 29 extend this to monoclonals of human origin and to encourage their wider use, are more helpful in describing techniques to reduce contamination and bring in to the equation the severity of the disease which is targeted. The wealth of information available on rodent antibodies makes any other choice for immunoaffinity chromatography far less attractive. The EEC draft guidelines 28 make no distinction between monoclonal antibodies for parenteral use and those used for the preparation of drugs (as for example for the preparation of an immunogen with therapeutic properties). They recommend that sources of animals and cell lines be well-defined and free from four specified viruses (Hantavirus, lymphocytic choriomeningitis virus, reovirus type 3 and Sendai virus) known to cause disease in

1

Column of lg~:~.[ MAb tO B ~,i.;;~1f

Co,umnof MAb to C

Contaminants

Pure A

Pure B

Pure C

A cascade system for the immunoaffinity purification of a number of products from one source.

man. Some 17 other viruses which, from our present state of knowledge, are regarded as less hazardous, are also identified and the guidelines require their absence from the final antibody preparation to be demonstrated. Account is also taken of the possibility of introducing virus from feeder cells commonly used in the development of the hybridoma or from the use of serum which may be necessary for tissue culture. The regulations also identify likely sources of protein contaminants that may prejudice efficacy in clinical use: if not chosen with care, the myeloma line may, for example, produce native immunoglobulins that contaminate the purified monoclonal antibody. Without specifying limits, the guidelines also emphasize the importance of minimizing contamination from DNA generally and from the hybridoma DNA specifically. On methods for preparing and exercising quality control on the pharmaceutical production of monoclonal antibodies, the USA draft guidelines 29 closely resemble those of the EEC but are confined explicitly to injectable antibodies. No reference is made to their use as reagents. However, if both health authorities are to be satisfied with a

projected use of a monoclonal antibody in an immunoaffinity column, the more exacting requirements of the EEC have to be taken into account. For this reason, some of the more specific recommendations of the USA guidelines, directed at i n - v i v o use are worth applying. In the tests for polynucleotide contamination they recommend fluorescence enhancement assays which allow the detection of nanogram amounts of nucleic acid and hybridization analysis using nick-translated hybridoma cell DNA to detect hybridoma DNA levels of below 10picograms m1-1. In Australia, the Commonwealth Department of Health 3° has followed the USA in confining their guidelines to monoclonal antibodies for therapeutic use implicitly excluding their use as reagents. These guidelines include useful practical techniques on viral decontamination using [%propiolactone which could be adapted to the treatment of immobilized monoclonal antibody for the purpose of immunoadsorption chromatography. If immobilization of the antibody improves stability as it does many proteins 31, the antibody in an immunoaffinity column may well survive decontamination procedures that inactivate soluble proteins and viruses. The most convincing evidence for the safety of drugs produced through the intervention of immunoaffinity chromatography would be proof of the absence of viruses likely to cause disease in man from the animals, cell-lines and animal products used in the preparation of the antibody 32'33 and the ability of decontamination procedures applied directly to a monoclonal antibody preparation or to an immunoaffinity column derived from it. The success or otherwise of a decontamination procedure can be demonstrated convincingly only in spiking experiments in which wellcharacterized viruses and nucleic acids are deliberately added to the antibody preparation and their inactivation or removal by nuclease treatment 34 demonstrated.

Conclusions The use of MAb-based IAC in the preparation of pharmaceuticals for

TIBTECH - APRIL 1987 [Vol. 5]

parenteral use, is still in its infancy but in the b r o a d e r field of p r o t e i n purification it has already b e e n p r o v e d a p o w e r f u l technique. Even w h e n the d e v e l o p m e n t costs of MAb are taken into account, IAC c o l u m n s are no m o r e e x p e n s i v e t h a n c o l u m n s for fast-flow liquid c h r o m a t o g r a p h y and can be u s e d r e p e a t e d l y w i t h o u t t i m e - c o n s u m i n g regenerations. T h e m a i n appeal of the t e c h n i q u e to the p r o t e i n purifier, h o w e v e r , lies in its ability to p r o d u c e a h o m o g e n e o u s protein in virtually a single step w i t h the prospects of a m u c h higher y i e l d t h a n is u s u a l l y obtained b y multistep c o n v e n t i o n a l methods. W h e n more e x p e r i e n c e has b e e n gained in IAC the special characteristics of MAb that are appropriate to this t e c h n i q u e m a y emerge and greatly simplify the d e v e l o p m e n t of n e w processes. With the benefit of these advantages and the increasing confid e n c e of Regulatory Authorities in the safety of MAbs, the t e c h n i q u e m a y well b e c o m e the m e t h o d of choice for the purification of therapeutic proteins.

References 1 Marrack, J. R. (1938) in The Chemistry of Antigens and Antibodies pp. 169-175, HMSO (London) 2 Heidelberger, M. (1939) Bact. Rev. 3, 49-95 3 Campbell, D. H., Luescher, E. and Lerman, L. S. (1951) Prec. Natl Acad. Sci. USA 37, 575-578 4 Day, E. D. (1966) Foundations of Immunochemistry pp. 162-171, Williams & Wilkins 5 Kohler, G. and Milstein, C. (1975) Nature 256, 495-497 6 Staehlin, T., Hobbs, D. S., Kung, H., Lai, C.-Y. and Pestkas, S. (1981) J. Biol. Chem. 256, 9750-9754 7 Nolli, M. L., Corti, A., Soffientini, A., Cassani, G. and Parenti, F. (1984) British Patent No. GB 84/22007

8 Bourgois, A., Delazay, M. and Fert, V. (1984) French Patent No. FR 84/ 14480

9 Le, J., Vilcek,

Barrowclough, B.S. and J. (1984) J. Immuno]. Methods 69, 61-70 10 Cohen, G. H. and Eisenberg, R.-J. (1986) World Patent No. WO 86/ O1517

11 "Hershberg, R. D. (1984) US Patent No. US 6229295

12 Yanagawa, S., Hirade, K., Ohnota, H., Sasaki, R., Chiba, H., Ueda, M. and

Goto, M. (1984) J. Biol. Chem. 259, 2707-2710 13 Heusser, C., Andreatta, R. H., Alkan, S. and Wood, J. (1984) Swiss Patent No. CH 1781/84

14 Smith, K. A. (1985) World Patent No. WO 85/01298

15 Reagan, M. E., Robb, M., Bornstein, I. and Niday, E. G. (1985) Thromb. Res. 40, 1-9 16 Church, W. R. and Mann, K.G. (1985) Thromb. Res. 38, 417-424 17 Chernausek, S. D., Chatelain, P.G., Svoboda, M.E., Underwood, L.E. and van Wyk, J.J. (1985) Biochem. Biophys. Res. Commun. 126, 282 18 Pierce, J. G. and Parsons, T. F. (1981) Annu. Rev. Biochem. 50, 456-495 19 Jack, G. W., Blazek, R., James, K., Boyd, J.E. and Micklem, L . R . J . Chem. Technol. Biotechnol. (in press) 20 Jack, G. W. and Blazek, R. J. Chem. Technol. Biotechnol. (in press) 21 Eveleigh, J. W. and Levy, D. E. (1977) J. Solid-Phase Biochem. 2, 45-78 22 Dawes, J. (1986) World Patent No. WO 86/OO91O

23 Liebman, H. A., Limentani, S.A., Furie, B. C. and Furie, B. (1985) Prec. Natl Acad. Sci. USA 82, 3879-3883 24 Hopp, T. P. et al. (1986) in Patentwatch, 6, 1, European Patent No. EPO 195680 25 Bazin, H. and Malache, J.-M. (1986) J. Immunol. Methods 88, 19-24 26 Chase, H. A. (1984) Chem. Eng. Sci. 39, 1099-1125 27 Eveleigh, J. W. (1982) Anal. Chem. Syrup. Set. 9, 293-303

28 Commission of the European Communities - Committee for Proprietary Medicinal Products (1986) Draft report on Requirements for the Production and Quality Control of Monoclonal Antibodies for Murine Origin Intended for Use in Man Draft

4, Rue de la Loi 200, B-1049 Brussels, Belgium 29 Federal Drug Administration (1985) Draft report on Points to Consider in the Manufacture of Injectable Monoclonal Antibody Products Intended for Human Use in Vivo, Office of

Biologics Research and Review Center for Drugs and Biologics, Bethesda, USA 30 Commonwealth Department of Health (1985) Draft report on Guidelines for the Production of Monoclona] Antibodies intended for Therapeutic use PO Box 100 Woden, ACT 2606

Canberra, Australia 31 Weetall, H. H. and Cooney, D.A. (1981) in Enzymes as Drugs (Holcenberg, J.S. and Roberts, J. eds), pp. 395-443, John Wiley 32 Levy, J. A., Lee, H.M., Kawahata, R.T. and Spit!er, L.E. (1984) Clin. Exp. Immuno]. 56,1.14-120 33 Carthew, P. (1986)J. Gen. Viro]. 67, 963-974 34 Beale, A. J. (1985) in Quo Vadis? Therapeutic Agents Produced by Genetic Engineering (Joueaux, A.,

Leygue, G., Moore, M., Roncucci, R. and Schmelck, P.H., eds), 'Quo Vadis?' Symposium, Sanofi Group, May 29-30, 1985, Tou]ouse-Labege, pp. 167-178, Medsi