Recent developments in downstream processing based on affinity interactions

TIBTECH - OCTOBER 1987 [Vol. 5]

3 Kelly, J. M. and Hynes, M. J. (1985) EMBO J. 4, 475-479 4 Hizeman, R. A., Hagie, F. E., Levine, H.L., Goeddel, D.V., Ammerer, G. and Hall, B.D. (1981) Nature 293, 717-722 5 Valenzuela, P., Medina, A., Rutter, W.J., Ammerer, G. and Hall, B.D. (1983) Nature 298, 347-350 6 Bitter, G. A. and Egan, K. M. (1984) Gene 32,263-274 7 Mellor, J., Dobson, M. J., Roberts, N.A., Tuite, M.F., Emtage, J.S., White, S., Lowe, P.A., Patel, T., Kingsman, A. J., and Kingsman, S. M. (1985) Gene 24, 1-14 8 Nunberg, J. H., Meade, J. H., Cole, G., Lawyer, F. C., MacCabe, P., Schwerckart, V., Tal, R., Wittman, V.P. Flatgaard, J. E. and Innis, M. A.,(1984) Mol. Cell. Biol. 4, 2306-2315 9 Innis, M. A., Holland, M. J., MacCabe, P. C., Cole, G. E., Wittman, V. P., Tal, R., Watt, K.W.K., Gelfand, D.H., Holland, J. P. and Meade, J. H. (1985) Science 228, 21-26 10 Boel, E., Hjort, I., Svensson, B., Norris, F., Norris, K. E. and Fiil, N. P. (1984) EMBO J. 3, 1097-1102 11 Ashikari, T., Nakamura, T., Tanaka, Y., Kiuchi, N., Shibano, Y., Tanaka, T., Amachi, T. and Yoshizumi, H. (1986) Agric. Biol. Chem. 50,957-964 12 Shoemaker, S., Schweickart, V., Ladner, M., Gelfand, D., Kwok, S., Myambo, K. and Innis, M. (1983) Bio/ Technology 1, 691-696 13 Shoemaker, S. P., Gelfand, D. H., Innis, M. A., Kwok, S.Y., Landmer, .M.B. and Schweickart, V. (1984) European

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Patent

Application

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Recent developments in downstream processing based on affinity interactions J. H. T. Luong, A. L. Nguyen and K. B. Male Novel purification processes have been developed, based on the interaction between complementary biomolecules, to circumvent the difficulties encountered by conventional affinity chromatography. Depending upon the procedure used for isolating the ligand-binder complex, the process can be termed affinity cross-flow filtration, affinity partition or affinity precipitation. This review describes the developments and potentials of such purification techniques. The c o m m e r c i a l realization of biot e c h n o l o g y will ultimately d e p e n d on the r e a d y availability of puriJ. Luong, A. Nguyen and K. Male are at the Riotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec, H4P 2R2, Canada.

fled e n z y m e s , proteins and other specialty chemicals. The bottleneck of c o m m e r c i a l i z a t i o n is not o n l y to p r o d u c e large quantities of heretofore rare p r o d u c t s but also to a p p r o a c h 100% in purity. Novel p r o d u c t s from r e c o m b i n a n t organisms are often unstable, especially in the p r e s e n c e of other similar materials. The dilute

~) 1987, Elsevier Publications, Cambridge 0166- 9430/97/$02.00

TIBTECH - OCTOBER 1987 [Vol. 5]

--Fig. 1

Mixture%

desired product is very sensitive to temperature, pH, shear, microbial contamination, and solvents used in purification. Such characteristics inevitably pose several technical problems on the design and operating conditions of the purification technique. Purification from complex biological mixtures has traditionally been performed by combining techniques which resolve substances according to differences in their overall physico-chemical properties 1. Extensive purification thus translates into a high yield loss and a high production cost. In many recombinant DNA bioprocesses, the purification of protein products can contribute up to 90% of the overall processing cost 2. New, robust purification procedures which possess both high resolution and recovery are still needed for very dilute and delicate products. This is the most critical area demanding innovative process development 3. For the last decade, impressive progress has been achieved in purification technology by exploiting the natural affinity displayed between biochemicals and their complementary ligands.

Affinity chromatography Affinity chromatography exploits the natural avidity displayed between biological molecules and their complementary ligands. This technique offers high selectivity, especially when monoclonal antibody (MAB) is used as the affinity ligand (immunoadsorbent chromatography). An affinity matrix can be prepared by chemically binding a complementary ligand, often via a suitable spacer molecule, to the surface of insoluble bead materials. Only the biomolecule that recognizes the immobile ligand will be retained on the matrix while other molecules pass by unretarded. Subsequently, a solution containing an appropriate eluant can be passed through the column to release and recover the bound molecule. Affinity chromatography, having revolutionized protein (enzyme) purification procedures in the 1970s, is one of the most widely used purification procedures. It can achieve a high degree of purification,

/

Macroliganid

IS)

/

,

Isolated Impurities Macroligand Product ligand-binder Idealized concept of affinity cross-flow filtration. (A) Separation of impurities, (B) Elution of product from ligand-binder complexes.

while providing good recovery yield. This method, however, is costly, laborious, and time-consuming since the affinity matrices are difficult to prepare, susceptible to degradation and only have a limited operating life. The system can only be operated batchwise and it uses a packed column that can become plugged and fouled. Consequently, pretreatment of the feed to remove solid contaminants is a prerequisite. The low throughput makes affinity chromatography a very low productivity system: its processing rate could be as low as 10 kg h -1 compared with that of precipitation procedures (>1000 kgh-1). Thus, it has been almost exclusively used for preparation of the very costly products such as urokinase 4, interferon, and antithrombin III. Affinity interactions can, however, be used rather more subtly: in affinity cross-flow filtration they can be used, in effect, to increase the molecular size of the desired substance: in affinity partitioning, they can be used to alter partition coefficients: and in affinity precipitation, they can be used to make the product precipitable under non-denaturing conditions.

Affinity cross-flow filtration Combining affinity interactions and membrane separation, a method called affinity cross-flow filtration has been developed 5. Its basic principle is that the substance to be purified (binder) when present in a crude mixture, will pass through the membrane. The binder, however, will be retained by the membrane w h e n it binds to a very high

molecular weight ligand (macroligand), while other unbound components of the mixture will pass through. The isolated ligand-binder complex is then treated with an appropriate eluant to desorb the binder from the ligand. The isolated binder is then collected and the ligand is recycled and reconditioned if necessary (Fig. 1).

Binding If a homogenate contains no particulate matter, a crude solution of the binder is mixed with the ligand and affinity binding takes place. Alternatively, in a more general approach which is applicable for continuous purification of material from a bioreactor, the crude solution and the macroligand are delivered separately to either side of a suitable molecular weight cut-off membrane. The binding occurs w h e n the substance to be purified passes through the membrane 5. As with other affinity methods, it is important to select a suitable elution procedure to keep conditions favorable for the biomolecules (P and L) and the membrane. After the elution step the macroligand is free and, after a reconditioning step to remove the buffer containing dissociating substances and/or competing ligands, it can be recycled. Reconditioning of the macroligand can be done in a separate ultrafiltration step.

Applications The affinity cross-flow filtration technique was applied to purify concanavalin A from a crude extract

TIBTECH- OCTOBER 1987 [Vol. 5] - - Table 1

Proteins and enzymes recovered by affinity cross-flow filtration Protein purified

Sources

Concanavalin A extract of

Alcohol dehydrogenase i~-galactosidase

Conavalia ensiformis S. cerevisiae extract

Escherichiacoli

Trypsin

mixture with chymotrypsin

Trypsin

mixture with chymotrypsin

Trypsin

mixture with chymotrypsin

Trypsin

pancreatic extract

Macroligand

% % Recovery Purity

heat-killed

70

ND

Ref. 6

Saccharomyces cerevisiae cells Cibacron Blue on starch modified agarose dextran-paminobenzamidine soybean trypsin inhibitor on dextran m-aminobenzamidine on polyacrylamide m-aminobenzamidine on polyacrylamide

ND

ND

5

70

ND

7

76

65

8

55

81

9

90

98

10,11

45

a

12

aContinuous process, purity verified by electrophoresis.

of Jack beans (Conavalia ensiformis) by using heat-killed cells of Saccharomyces cerevisiaeas the affinity adsorbent 6. Using n-glucose (0.8 M) as the eluant, a highly purified product was obtained at an overall yield of 70%. The affinity interaction between concanavalin A (mol. wt = 102 000) and heat-killed cells (mean diameter of 5 ~m) occurred in a mixing chamber, washing and dissociation were performed in membrane units with molecular weight cut:off of 300-1000 kDa. Consequently, concanavalin A in free form passed through the membrane. However, the concanavalin A - heat killed cells complex, was large enough and could not permeate. Affinity cross-flow filtration was also applied to purify alcohol dehydrogenase (ADH) from a crude extract of S. cerevisiae 5. Cibacron Blue, a dye that can bind to NAD- and ATPrequiring enzymes, was immobilized to starch granules to form a macroligand. After centrifugation to remove all particulate matters, the yeast homogenate was mixed with the macroligand. In this process, a hollow-fiber with a molecular weight cut-off of 500000 was used to isolate the macroligand-binder complex which was then dissociated with potassium phosphate solution (0.55 M). Neither product purity nor yield were reported in this work. This concept was recently extended to purify [3-galactosidase from cell homogenates of Escherichia

coli 7. The affinity beads used were the commercially available p-aminobenzyl- 1-thio-[3-D-galactopyranoside agarose. The process consists of two identical stages of absorbing and desorbing where the affinity beads were kept in the external compartments of two containers equipped with concentric cylindrical filtration devices (20 ~m membrane filter). The feed stream containing the crude ~-galactosidase and the wash buffer were mixed and fed to the adsorption unit as one stream. The resulting affinity adsorbent-binder complex was then transferred to the desorbing stage where dissociation took place at pH 10. The containers were agitated on a rotary shaker in order to keep the affinity beads in suspension. The suspension of affinity absorbent was recirculated between the external compartments of the two containers by a multichannel peristaltic pump. The recovery yield of this continuous purification process was reported to be 70% with a corresponding productivity of 25.2 U(ml gel) -1 h -1. Limitations and prospects Affinity cross-flow filtration appears to be limited by the availability of suitable adsorbents. Furthermore, when particle-bound ligands are used, there is a limit to the amount of binder that can be bound. This is well recognized from experiments in conventional affinity chromatography. Such a limitation could be overcome by 'tailoring' the water-

soluble macroligand. For instance, attempts 8'9 to separate trypsin from chymotrypsin using p-aminobenzaminidine linked to dextran, were not encouraging: dextran seemed to bind chymotrypsin non-specifically. A highly specific macroligand which w o u l d substantially increase the retention of the species to be purified was needed. Recently, Luong eta/. 1°'11 synthesized a high molecular weight watersoluble macroligand (> 100 kDa) having an acrylamide backbone with maminobenzamidine groups as pendants. Its trypsin-binding capacity approached the theoretical value of 120:1 (weight of bound trypsin: weight of aminobenzamidine present) which was considerably higher than that of insoluble gel matrices. Moreover, the homogeneous character of the system facilitated the ligand-binder interaction so that the binding process occurred almost instantaneously. The trypsin-ligand complex could be easily dissociated by arginine (0.5 M). In batch, the procedure could purify trypsin from a trypsin-chymotrypsin mixture with 90% yield and a purity of 98%. Operating continuously, and using benzamidine as the eluant because of its specificity, an overall yield of 45% of highly purified trypsin from porcine pancreatic extract was accomplished. Literature reports on affinity cross-flow filtration can be found in Table 1. The high resolution and recovery possible with affinity cross-flow filtration, along with its capability of processing unclarified and viscous liquids, have given a large impetus to developing its application further. The technique can be applied for purification of biochemicals from the liquid immediately after completion of fermentation. In developing a particular process, attachment of the ligand to suitable macromolecules, is not a problem and can be accomplished by many well-known procedures. The choice of ligand is important, since its properties other than its essential specificity will affect the efficiency and operating life of the system. The insensitivity of chemical ligands to the shear force, temperature, pH, etc., means they are easy to handle and possess a long operating life. However, suitably

TIBTECH - OCTOBER 1987 [Vol. 5]

specific chemical ligands are not always available in which case, biochemical affinity, e.g. antibodyantigen interaction, can be exploited. However, handling difficulty may arise. Presently, both types of ligands are subjects of intensive investigations. The technological limit to the use of affinity cross-flow filtration has been the lack of suitable membranes in terms of molecular weight cut-offs, flux, operating life, etc. Today, however, the membranes are produced with high resistance to acids, bases, alcohols, and temperatures, and which allow very effective cleaning. Possibilities also exist for fabricating membranes with high molecular weight cut-offs between 1 0 5 t o 106, a region of great interest for protein separations (Table 2). For a successful development of affinity cross-flow filtration, the semi-permeable membrane should exhibit: • high hydraulic permeability to solvent (water); • sharp molecular weight cut-off; • good mechanical durability, chemical and thermal stability; • high fouling resistance; • easy cleaning and sterilization; • long life. It should be noted that fouling, to a certain extent, is still one of the bottlenecks of membrane technology. The interaction between the

macroligand and the membrane is another important matter. The absorption of the macroligand to the membrane could result in a significant reduction in the permeation rate and may form a dynamic membrane with its own rejection characteristic. This problem could be circumvented by adding electrostatic charge to the membrane~and/or the macroligand. This is an important and fascinating area in separation, a subject to which greater research effort should be devoted. It is likely that this promising technique will be used very extensively in the not-too-distant-future, at least at small production scales.

Affinity partition The partition of proteins or enzymes between the liquid phases of aqueous two-phase systems such as dextran and polyethylene glycol (PEG), PEG and a salt etc., is a wellknown phenomenon. Extractions of enzymes using liquid-liquid two phase systems have been carried out in a very large scale, up to 3001, without any technical problems 13. It is noteworthy that the development of largescale enzyme extraction processes has been limited so far to PEG/ dextran and PEG/salt systems. The following reasons have contributed to this circumstance: the general applicability of the system, the

relative suitability of its physicochemical properties, its biodegradability, and its non-toxicity. Success with phase systems depends on the ability to manipulate phase composition so as to obtain appropriate partition coefficients for the materials of interest. To a certain extent, phase systems can be manipulated by choice of polymers, polymer molecular weight, and polymer concentration 14. The selectivity of aqueous two phase systems can also be greatly improved by using polymer-bound affinity ligands. In affinity partition, the ligand is bound to one of the phase-forming polymers. Consequently, the modified polymer controis the partition of proteins or enzymes in the phase system and thus leads to the accumulation of such materials in the phase containing affinity ligands. Recovery of the product and recycling of the polymer-bound ligands are also of importance. Addition of potassium phosphate to the separated modified-PEG phase will, in general, dissociate the ligandbinder interaction as well as inducing the formation of a new two phase system. The product usually favors the salt-rich phase and it can be recovered by diafiltration. The ligand-bound polymer-rich phase contains an appreciable amount of salt and it must be diafiltered to

--Table 2

Commercially available ultrafiltration membranes Manufacturer Amicon Corp.

Abcor, Inc.

Millipore

Dorr-Oliver Romicon, Inc. Ultra-Pore, Inc. (Nucleopore)

N/A, not available.

Membrane designation XM 100 HP 100 XM 300 MSD-400 MSD-403 PTHK PSVP PTMK D-300 GM-80 C 100 F 100 C 300 F 500 F 100

Mol. w t cut-off 100 100 300 100 250

000 000 000 000 000

100 1 000 1 000 300 80 100 100 300 500 1 000

000 000 000 000 000 000 000 000 000 000

Chemical composition Acrylic Polysulfone Acrylic Proprietary Noncellulosic polymers Polysulfone Cellulose acetate Polysulfone Dynel Noncellulosic Proprietary

pH l i m i t

Max. t e m p . (°C)

Max. p r e s s u r e (psi)

N/A 1.5-13 N/A 0.5-13 0.5-13

70 50-70 70 90 90

25 25 25 150 150

1-14 2-8 1-14 2-12 1.5-13 N/A

50 50 90 60 45 N/A

100 200 100 N/A 25 N/A

TIBTECH - OCTOBER 1987 [Vol. 5]

- - T a b l e 3'

Proteins or enzymes recovered by affinity partitioning Protein purified

%

Sources

Trypsin a

mixture with chymotrypsin Serum albumin a plasma Oxosteroid extract from isomerase a culture of

Ligand

%

Recovery P u r i t y Ref.

p-amino-

92

76

16

benzamidine palmitate estradiol

91 37

~100 ND

17 19

lecithin

ND

ND

20

ND

17

Pseudomonas testosteroni Colipase96 a Glucose-6phosphate dehydrogenase b

tryptic hyd rolysate of colipaselol yeast extract

Cibacron Blue

ND, not determined. abatch processing, recovery and purity reported for one step. bcontinuous processing.

quite useful for attaching a variety of affinity ligands. Several PEG derivatives are also available for use in affinity partitioning: PEG monopalmitate, PEG octadecyl ether, Ethylenediamine PEG, PEG-protein, PEG-Cibacron Blue, Trimethylamino PEG and PEG-Sulfonate 24. As with affinity cross-flow filtration, the choice between the biological ligand and the chemical ligand is delicate. Undoubtedly, the cost and the quality of the purified product will dictate the type of ligand used.

Affinity precipitation recycle the ligand-bound polymer. Solvent extraction is another possibility which may be used to extract the ligand-bound polymer. For instance, Kopperschl~ger and Johansson 15 recovered the polymeric affinity ligand by chloroform extraction. The recovery of the modified soluble polymer, however, is still tedious and problematic and may be too expensive to be used on a technical scale. Affinity partitioning of trypsin with p-aminobenzamidine as the polyethylene glycol-bound ligand was first attempted by Takerkart et el. 16. In dextran-polyethylene glycol systems, PEG is essentially contained in the upper phase. In such systems, recovery of trypsin activity in the upper phase was only 40% of the total activity (10-4M of trypsin was introduced to the system). By attaching p-aminobenzamidine, a strong inhibitor of trypsin, to PEG, the recovery of trypsin activity in the upper phase could be increased to 92% of the total activity. The extraction of serum albumin with PEG-bound palmitic acid was another application of affinity partition 17. By introducing human serum to such a two phase system, about 90% of albumin was bound to palmitate moieties in the PEG phase while other proteins were partitioned totally in the dextran phase. An elegant application of affinity partition was reported using PEGbound Cibacron Blue F3G-A or Procion Red HE-3B and dextran to form an aqueous two phase system TM. This system was used for purification of glucose-6-phosphate dehydrogenase with a yield of about 95%.

Polymer-bound Cibacron Blue F3GAnother promising approach A was also exploited for the large- which involves the precipitation scale purification of phosphofructo- of the ligand-product complex is kinase from baker's yeast. Affinity defined as affinity precipitation. partitioning in an aqueous two-phase The process makes use of bisystem containing Cibacron Blue functional ligands such as nucleoPEG, yielded a 58-fold purification of tides connected by a spacer (Bisthe enzyme in 67% overall yield NAD+). Such bifunctional ligands within 3 hours. Literature reports will bind to the active sites on on affinity partitioning of enzyme/ multi-subunit proteins, thus nonproteins can be found in Table 3. covalently cross-linking them and Continuous purification of en- effecting precipitation. The first inzymes by aqueous two phases has vestigation of affinity precipitation 25 been demonstrated. An example of utilized a bifunctional nucleotide this application is the purification of derivative, N2,NE-adipohydrazidofumarase from Brevibacterium am- bis-(N6-carbonylmethyl-NAD) (Bism o n i a g e n e s by a two-step process NAD) to precipitate the tetrametric developed by Hustedt et el. 21 where enzyme lactate dehydrogenase PEG and salt were used to form a (LDH). liquid-liquid two phase system. The explanation of the precipitaIn view of this, it is likely that the tion mechanism is that LDH, Bisaffinity partitioning process can also NAD and pyruvate (LDH was disbe operated continuously. solved in 0.05 M phosphate buffer (pH 7.5), 0.1M with respect to Limitations a n d p r o s p e c t s pyruvate) form strong dead-end The major drawback of affinity ternary complexes, and since LDH is partition is the cost of dextran. a tetrameric enzyme, Bis-NAD Although crude dextran (inexpen- can interact with two LDH molsive) could be used as a phase ecules to form large aggregates. forming constituent, its high mol- The solution eventually becomes ecular weight results in a highly insoluble w h e n these aggregates viscous medium: fractionated dex- are sufficiently large. NADH (10 raM) tran (low molecular weight) has was added to the separated premanageable viscosities, but it is too cipitate to dissociate the enzyme expensive. Less expensive substi- from the affinity ligand. Gel filtration tutes such as starch derivatives, or was used to remove the enzyme from pullulan 22'23 are commercially avail- pyruvate and Bis-NAD as a final able at a lower price. step. The recovery yield reported For affinity partitioning to reach its was 85%. Affinity precipitation was full potential simple methods must also utilized to isolate erythrobe available for attaching the ligand cytes with N,N'-bis-3-(dihydroxyto the polymer. A few dextran borylbenzene)-adipamide (Bis-pBA). derivatives such as octadecylamino This ligand probably displays the dextran, octadecylamide dextran, lectin-like property of agglutinating and N-amino dextran should be red blood cells 26.

TIBTECH- OCTOBER1987[VoL5]

Undoubtedly, affinity precipitation could be extended to purify enzymes/proteins other than dehydrogenases if suitable bifunctional ligands were available. Noteworthy is the fact that besides dead-end complex formation, other ternary complexes could be developed, e.g. complexes with coenzyme and inhibitor, or with coenzyme-substrate adducts 2°. The formation of ternary complexes is particularly useful w h e n the interaction between the bifunctional ligand and the enzyme is not sufficiently strong. Another promising approach developed by Schneider e t a ] . 24 involves the precipitation of the ligand-product complex (Fig. 2). For purification of trypsin from bovine pancreas, a water-soluble polymer polyacrylamide was developed which bore a ligand group (paminobenzamidine), and a precipitation group (benzoic acid) which permitted a quantitative precipitation of the affinity polymer. The polymer was added directly to a crude extract under conditions favoring the binding of the desired protein and was then precipitated. After removal of the supernatant, the protein of interest was eluted from the polymer and the polymer recycled. The recovery yield was 90% and the recovered trypsin was essen-

--Fig. 2

(A) _~

(B)

(C)

_

,*-macroligand

1

0

~t t~ * /*-crude extract

J ~ j*

0 *-supernatant _ _C] __ (impurities)

~

,

~

-I

~-Iipg::idp~r:duct

•

*-product

Idealized concept of affinity precipitation. (A) Binding to macroligand, (B) Precipitation of ligandbinder complexes to be followed by supernatant removal, (C) Dissociation of product form macroligand.

tially pure and active. In this process, the amount of polymer added to the medium was very low (0.1-0.5%). Thus, non-specific adsorption of proteins on the polymer was also low. The polymer could be recycled for repeated use without losing its binding efficiency. The average polymer loss per cycle was about 1%. The method promises much since precipitation and recovery of the precipitate are classical procedures which can be applied to large volumes. Limitations a n d prospects Affinity precipitation has been demonstrated as a powerful technique for preparative enzyme purification. This technique offers the most economical use of the immobilized ligand for rapid and specific isolation of proteins on a large scale. It seems possible that affinity precipitation will also find considerable application in the large-scale purification of proteins and other specialty chemicals but the development, to date, of this technique has been slow compared to that of affinity cross-flow filtration and affinity partitioning.

Conclusion The unique specificity and reversibility of biological interactions have opened a new horizon for the development of purification technologies. In their qualities of scale, resolution, recovery yield, and capacity, the techniques based on affinity interactions have the potential to replace existing process technologies in large-scale protein (or enzyme) fractionation such as conventional affinity chromatography and high performance liquid affinity chromatography (HPLC). We will end by quoting Genentech, the world's leading biotechnology company: 'Laboratory creation of a new product is the first step. It only begins to count when the product is purified and packaged'. References 1 Lowe, C. R. (1984) J. Biotechnol. 1, 312 2 Dwyer, J. L. (1984)Bio/Technology, 2, 957-964 3 Michaels, A. S. (1984) Chem. Eng. Pros. 80, 19-26 4 Herion, P. and Bohlen, A. (1983)

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