Affinity chromatography in the industrial purification of

J. Biochem. Biophys. Methods 49 Ž2001. 575–586 www.elsevier.comrlocaterjbbm

Review

Affinity chromatography in the industrial purification of plasma proteins for therapeutic use Thierry Burnouf ) , Mirjana Radosevich Human Plasma Product SerÕices, 18 Rue Saint-Jacques, 59800 Lille, France

Abstract Affinity chromatography is a powerful technique for the purification of many proteins in human plasma. Applications cover the isolation of proteins for research purposes but also, to a large extent, for the production of therapeutic products. In industrial plasma fractionation, affinity chromatography has been found to be particularly advantageous for fine and rapid capture of plasma proteins from industrial plasma fractions pre-purified by ethanol fractionation or by ion-exchange chromatography. To date, affinity chromatography is being used in the production of various licensed therapeutic plasma products, such as the concentrates of Factor VIII, Factor IX, von Willebrand Factor, Protein C, Antithrombin III, and Factor XI. Most commonly used ligands are heparin, gelatin, murine antibodies, and, to a lesser extent, Cu2q. Possible development of the use of affinity chromatography in industrial plasma fractionation should be associated to the current development of phage display and combinatorial chemistry. Both approaches may lead to the development of tailor-made synthetic ligands that would allow implementation of protein capture technology, providing improved productivity and yield for plasma products. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Affinity chromatography; Industrial purification; Plasma proteins; Therapeutic use

1. Introduction Human blood is a very unique biological material comprising both cellular elements Žred cells, platelets, lymphocytes. and plasma. Red cells lymphocytes and plasma can be used as single donor products but can also be pooled and processed industrially to prepare a large range of therapeutic products. Many therapeutic products can be extracted from human blood. Although a few products can be made from red blood cells Žhemoglobin. or leucocytes Žalpha-interferon., )

Corresponding author. Fax: q33-3-2838-1935. E-mail address: [email protected] ŽT. Burnouf..

0165-022Xr01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 2 X Ž 0 1 . 0 0 2 2 1 - 4

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T. Burnouf, M. RadoseÕichr J. Biochem. Biophys. Methods 49 (2001) 575–586

essentially all pooled blood products are derived from plasma. Currently, over 20 distinct licensed plasma-derived protein products are already available for clinical use. Plasma fractionation is therefore a ‘cracking process’ where production processes of the various derivatives are closely integrated. Although the traditional plasma fractionation methods are based on ethanol fractionation, more and more, plasma fractionation technologies include chromatographic steps w1,2x. Affinity chromatography has been introduced many years ago for the industrial extraction of plasma Antithrombin III, and other, more recent, significant applications have included the purification of Factor VIII, von WillebrandrFactor VIII complex, Factor IX, and Protein C. Following an earlier review w3x, this paper provides an update on the role of bioaffinity in the purification and production of plasma proteins and attempts to anticipate what could be future development in this area, considering the growing needs of the market for plasma products. 2. Affinity chromatography in industrial plasma fractionation Ethanol fractionation, introduced in the mid-1940s for the extraction of immunoglobulin and albumin from plasma w4,5x, and cryoprecipitation are still the major technologies used in most plasma fractionation plants w6x. Although this might sound surprising, this illustrates the soundness of the technology as well as its established safety andror industrial advantages w7,8x. However, column chromatography, integrated with cryoprecipitation and ethanol fractionation of plasma, has demonstrated to be an excellent industrial adjunct for the purification of coagulation factors and protease inhibitors w9,10x. The introduction of those production methods has dramatically improved the purity of plasma products, especially coagulation factors. As a benefit, the common side effects Žhemolysis, hypotension, fever. associated with the clinical use of the early generation of plasma products have become very rare. Table 1 summarizes the most current plasma fractionation methods used for some of the most important licensed plasma products. As outlined below, affinity chromatography plays an important, but variable, role in the manufacture of the various classes of plasma products: coagulation factors, protease inhibitors, anti-coagulants, albumin, and immunoglobulins. 2.1. Coagulation factors 2.1.1. Factor VIII All current licensed plasma-derived Factor VIII concentrates are produced from cryoprecipitate, a precipitate that is obtained by thawing a pool of human plasma Žfrom 1000 to 4000 l. at a temperature between 1 and 3 8C. Traditionally, Factor VIII concentrates were manufactured by precipitation methods. Chromatography has been introduced in the manufacturing process of Factor VIII concentrates in the late 1980s w11x. Anion-exchange and affinity chromatography, and to a lesser extent size-exclusion and immobilized heparin affinity chromatography w9x, are now commonly used for Factor VIII purification, sometimes in combination. Immobilized murine monoclonal antibodies directed towards human Factor VIII coagulant proteins or von Willebrand Factor, the physiological carrier of Factor VIII, are


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Table 1 Overview of plasma fractionation methods Licensed plasma products

Traditional fractionation emethod

Common industrial chromatographic processes

Factor VIII

Cryo-precipitationq precipitation

Factor IX

DEAE adsorption of cryo-poor plasma

Von Willebrand Factor Factor XI

Cryo-precipitationq precipitation NAa

Fibronectin

NAa

Antithrombin III

NAa

Alpha 1-antitrypsin

NAa

Protein C

NAa

Albumin

Ethanol fractionation

Immunoglobulin G

Ethanol fractionation

Anion-exchange chromatography w11x Immunoaffinityqion exchange chromatography w12–14x Anion-exchangeqimmobilized heparin affinity chromatography w17–21x Anion-exchange chromatographyq immunoaffinity w22,23x Anion-exchangeqCu Metal-chelateaffinity chromatography w24x Anion-exchangeqimmobilized gelatin affinity chromatography w26,27x Anion-exchangeqimmobilized heparin affinity chromatography w31x Anion-exchangeqcation-exchange chromatography w32x immobilized gelatin affinity chromatography w27x immobilized gelatinqimmobilized heparin w30x affinity chromatography Immobilized heparin affinity chromatography w33x Anion-exchange chromatography w36x Anion-exchangeqsize exclusion chromatography w37x Anion-exchangeqimmobilized heparin affinity chromatography w3x Anion-exchangeqImmunoaffinity chromatography w43x Ethanol fractionationqanion-exchange chromatography w8x Anion-exchangeqcation-exchangeq size-exclusion chromatography w47x Ethanol fractionationqion-exchange chromatography w55x Anion-exchangeqimmobilized arginin affinity chromatography w56x

a

Not applicable.

the major ligands used to make current immuno-affinity purified Factor VIII products w12–14x. A crude cryoprecipitate extract is injected into the monoclonal antibody column. Factor VIII elution is achieved by 40% ethylene glycol w13x or calcium to dissociate Factor VIII from von Willebrand Factor w14x. Finishing on aminohexyl agarose or QAE eliminates possible murine monoclonal antibodies or trace murine components leaching from the immuno-adsorption column. Direct affinity chromatography capture of Factor VIII from plasma on immobilized dimethylamino-propylcarbamylpentyl-Sepharose CL-4B has been evaluated experimen-

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tally w15x. However, the difficulty in recovering other plasma fractions has not allowed further industrial development. Immobilized lentil lectin has been described for the purification of the Factor VIIIrvon Willebrand Factor complex w16x but there has been no industrial application. 2.1.2. Factor IX All industrial high-purity plasma-derived Factor IX concentrates are obtained by chromatographic purification from cryo-poor plasma Žthe supernatant obtained after the cryoprecipitate is removed.. In most cases, production processes involve the use of the following affinity ligands: heparin w17–21x, murine anti-human Factor IX antibodies w22,23x, or metal chelate w24x; sulfated dextran w25x has also been described. Such processes yield a Factor IX concentrate with a specific activity above 150 IUrmg. Processes usually involve capture of the Factor IX complex Žcontaining Factors II, VII, X, and IX, Protein C, Protein S and other proteins. from cryo-poor plasma on DEAE Sephadex A-50 or DEAE cellulose. The DEAE-Sephadex eluate is then either directly chromatographed on sulfated dextran, providing isolated fractions in a single step w25x, or, more often, further purified on DEAE and an immobilized-heparin column w17–19x. Factor IX can also be extracted from cryo-poor plasma by DEAE-Sepharose, heparin-Sepharose, and a cation exchanger w20x or a combination of hydrophobic-interaction chromatography with immobilized heparin w21x. As for Factor VIII, immunoaffinity is also used for large-scale purification of Factor IX w22,23x. The cryo-poor plasma is purified by DEAE-Sephadex, followed by immunoaffinity, and polished by aminohexyl-Sepharose to eliminate residual murine antibodies w22x. In another approach, cryo-poor plasma is pre-purified on DEAESpherodex, and Factor IX is further isolated by immunoaffinity chromatography and polished by DEAE-Sepharose to remove anti-Factor IX antibodies w23x. Immobilized metal-chelate affinity chromatography ŽIMAC. has recently been introduced in a large-scale manufacturing process of Factor IX w24x, representing the first example of its industrial application in plasma fractionation. A DEAE-Sepharose eluate is loaded onto a column of Cu2q Sepharose at high ionic strength, under conditions where prothrombin does not bind. Factor X is eluted with a buffer at low ionic strength and low pH. Protein C and Factor IX are subsequently eluted with glycine-containing buffers. 2.1.3. Von Willebrand Factor An industrial high-purity von Willebrand Factor preparation, with a specific activity of about 150 IUrmg is obtained from cryoprecipitate by two anion-exchange-chromatographic steps on DEAE-Toyopearl 650 M and affinity chromatography on immobilized gelatin w26x. The gelatin step serves to remove fibronectin that tends to be eluted together with von Willebrand Factor on the anion exchanger w27x. Von Willebrand Factor does not bind to immobilized gelatin, and is recovered in the breakthrough fraction, while fibronectin is adsorbed on the resin. 2.1.4. Fibronectin Affinity chromatography on immobilized gelatin is the standard approach to the purification of fibronectin, although immunoaffinity on anti-fibronectin monoclonal


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antibody has also been evaluated experimentally w28,29x. A two-step chromatographic process combining immobilized gelatin and immobilized heparin adsorption, has been described as a way to improve product purity w30x. There are no licensed fibronectin products yet, so immobilized gelatin remains of a limited use to-date for obtaining fibronectin preparations. 2.1.5. Factor XI An industrial Factor XI concentrate is obtained by chromatography of cryo-poor plasma on DEAE-cellulose and heparin-Sepharose, following a purification strategy that is similar to that used to extract a therapeutic antithrombin III concentrate w31x. Purification of Factor XI on the heparin column involves a 0.35 M NaCl wash, followed by desorption with 2 M NaCl w2x. Another industrial process combines anion and cation exchange w32x. 2.2. Protease inhibitors 2.2.1. Antithrombin III Antithrombin III was the first example of a plasma protein industrially extracted by affinity chromatography. This was achieved by exploiting the ability of Antithrombin III to bind to heparin, its physiological co-factor w33x. Immobilized-heparin chromatography is still the standard technique for the purification of Antithrombin III from human plasma. In one purification approach, cryo-poor plasma is run through a DEAE ion-exchanger, to capture Factor IX, then on immobilized heparin under conditions Žphosphate buffer at close to neutral pH and low ionic strength. that bind Antithrombin III. The gel is washed with a buffer, containing 0.25 to 0.35 M NaCl, and Antithrombin III is eluted with 0.45 M NaCl or higher salt concentration w31,34x. An alternative affinity-chromatographic process on immobilized heparin uses Cohn ethanol precipitate IV-1 as the starting plasma material w9x. Some manufacturers have introduced a process comprising double binding of antithrombin III on immobilized heparin. When viral inactivation of purified Antithrombin III is achieved by heat treatment at 60 8C for 10 h Žpasteurization., there might be some degree of protein denaturation. Therefore, the second adsorption step on immobilized heparin, performed after the pasteurization step, allows a selective elimination of Antithrombin III molecules having lost their capacity to bind heparin w35x. 2.2.2. Alpha 1-antitrypsin Therapeutic alpha 1-antitrypsin preparations are obtained by processes that combine ethanol fractionation and ion-exchange and size-exclusion chromatography w36,37x. Affinity chromatography is not used in the manufacture of the licensed products. Laboratory-scale work has revealed the potential of affinity chromatography on immobilized concanavalin w38x, or Cibacron Blue or Procion Red w39,40x to facilitate the purification of alpha 1-antitrypsin from plasma fractions containing albumin, which share a number of biochemical features, in particular, molecular size and isoelectric

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point. As alpha 1-antitrypsin contains reactive thiol groups, thiol-disulfide-exchange chromatography of plasma, treated with 0.02 M 2-mercaptoethanol, has also been described for laboratory-scale purification of alpha 1-antitrypsin w41x. All these approaches are inapplicable to large-scale processing because of, e.g. risks of ligand leakage or difficulties in integration with the whole plasma fractionation scheme. 2.2.3. Inter-alpha-trypsin inhibitor A process has been described for the purification of inter-alpha-trypsin-inhibitor from cryo-poor plasma w42x. The purification approach is based on the combination of two anion-exchange-chromatographic steps, followed by adsorption on immobilized heparin. The first anion exchanger, DEAE-Sephadex A-50, allows the capture of inter-alphatrypsin inhibitor, together with the Factor IX complex, from cryo-poor plasma, while the second anion exchanger, DEAE-Sepharose Fast-Flow, allows separation from Factor VII and Factor IX. Immobilized heparin is designed to remove the complement component C4, Factor X, and Protein C that are eluted together with inter-alpha-trypsin inhibitor from the DEAE-Sepharose Fast-Flow column. 2.2.4. Other protease inhibitors Therapeutic C1-inhibitor is also prepared from human plasma. There is, however, no report on the use of affinity chromatography for the manufacture of licensed products. 2.3. Anticoagulants Affinity chromatography is used to prepare therapeutic plasma-derived Protein C concentrates. One process involves chromatography of cryo-poor plasma on DEAE Sephadex A50, to capture Protein C together with the Factor IX complex, DEAE-Sepharose FF, to separate Factors II, VII, and IX, followed by DMAE-Fractogel EMD and heparin-Sepharose, to ensure final polishing of the Protein C eluate w3x. Another approach is based on immunoadsorption on immobilized murine anti-human Protein C antibodies. w43x. The last method combines two ion-exchange steps and the immunoaffinity column. The use of a calcium-dependent monoclonal antibody directed against the gamma-carboxyglutamic acid domain of the light chain of Protein C, has also been described w44x. Based on the fact that Protein C has 12 surface-accessible histidines that can play a role in metal binding, a laboratory-scale technique based on immobilized Cu2q metal-affinity chromatography of Cohn Fraction IV-1 has been developed w45,46x, but it is not yet used at an industrial scale to make a licensed concentrate of Protein C. 2.4. Albumin Most therapeutic albumin products are obtained by ethanol fractionation of human plasma, following the technology that was introduced in the 1940s w8x. Processes combining ethanol fractionation and chromatography are also being used, where one chromatographic step, generally an anion-exchange chromatography, is used to polish the albumin fraction in order to remove contaminants, including vaso-active substances, albumin polymers, or endotoxins. A process combining one ethanol fractionation step


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with a three-step chromatographic procedure, anion-exchange, cation-exchange, and size-exclusion chromatography, is being used industrially w47x. No industrial process uses affinity chromatography to extract plasma albumin. This may have to do with the fact that albumin is present in relatively high concentration in plasma Žabout 40 grl., therefore making a direct capture of albumin from crude plasma industrially complex. However, attempts to develop affinity chromatography have been made in the past. Immobilized dye molecules, in particular Cibacron Blue 3GA, a blue anthraquinone dye, containing a monochlorotriazine-reactive group, could be very effective for purifying albumin at the research and pilot-plant scales. Triazine-dye affinity chromatography on immobilized Cibacron Blue F3GA has been used on a pilot-scale to purify albumin, obtained by a complete chromatographic procedure, starting with cryo-poor plasma w48x. Cibacron Blue F3GA-Sepharose Žor Procion Blue Sepharose CL6B. has also been described for the recovery of albumin from Cohn ethanol precipitate IV4 w49,50x. These processes have been scaled up to 40-l column to bind 1.1 kg of albumin with a recovery from Fraction IV4 of more than 98%. The final product met Pharmacopeia quality requirements but was not developed further for therapeutic application due to regulatory constraints Žsafety and toxicity issues associated with the use of Cibacron Blue.. The heterogeneity and low purity of the Cibacron dye w51x have been obstacles to its introduction in the purification of albumin at production scale for clinical use, but the methodology may be applied for producing albumin as a component of cell culture medium. Cibacron Blue 3GA has been shown to have the capacity to bind more than 99.5% of hepatitis B particles from plasma protein solution, suggesting the possible risk of elution of viruses together with the albumin fraction w52x. This factor may have also discouraged further attempts to apply the methodology to the manufacture of a therapeutic product. Alternative approaches based on affinity chromatography of albumin have been described but have not found practical applications on an industrial scale. Ligands evaluated include immobilized bilirubin, fatty acids, and dyes. Immobilized-bilirubin affinity chromatography w53x showed poor specificity and low capacity, and the ligand decomposed in light. Immobilized alkyl derivatives of succinic acid have yielded albumin w54x with a relatively low recovery of 60% upon elution with octanoate. Considering that albumin is the major plasma protein and that the backbone of plasma fractionation technology is highly dependent on the albumin purification process used, any change in the upstream purification process of albumin would require revalidation of the purification techniques used for most other plasma products. Since ethanol fractionation Žwhich can be combined with terminal anion-exchanger polishing, to gain from the higher purification factor afforded by chromatography. is considered to provide albumin preparations with good recovery from plasma and acceptable quality profiles for therapeutic applications, it is unlikely that affinity-based albumin purification processes will be widely implemented upstream of the plasma fractionation process in the near future. Nevertheless, some alternative uses of albumin, e.g. as a stabilizer of recombinant products or a culture-medium substitute, may prompt the development of refined, affinity-based polishing purification techniques, yielding ultra-pure albumin from Cohn supernatant fraction IV or Precipitate V.

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2.5. Immunoglobulins G Traditionally, immunoglobulin G preparations have been produced exclusively by precipitation techniques, predominantly ethanol fractionation. In recent years, however, chromatographic polishing steps have been introduced downstream of the ethanol fractionation steps, to remove protein contaminants which were associated with clinical side-effects, or to eliminate chemicals used during solvent detergent viral inactivation w55x. Such polishing steps are usually based on ion-exchange chromatography or C18 adsorption. There is very limited industrial application of affinity chromatography, either as a mainstream purification tool or as a polishing step, for the current IgG preparations for clinical use. However, a pilot-scale chromatographic purification process of IgG from cryo-supernatant, involving an arginine Sepharose 4B adsorption step, has been described recently w56x, yielding a product meeting Pharmacopeia requirements. Affinity chromatography on Protein A or Protein G, are not used in industrial plasma fractionation, probably because of cost issues and risks of leaching, and also because of the large quantity of gel that would be required to purify the tons of IgG produced by the major plasma fractionators. 2.6. Other proteins Transthyretin has been purified by affinity chromatography on immobilized Remazol Yellow GGL w57x, and an experimental production procedure for obtaining a 80% pure product from plasma supernatant II q III has been developed w58x. The immobilized lectin jacalin has been found to bind plasma proteins bearing O-linked oligosaccharides, including IgA, C1-inhibitor, and plasminogen w59x. There has been no industrial application of such a purification approach for therapeutic products. A synthetic ligand, obtained by combinatorial chemistry, has been found to bind IgG as well as IgA and IgM w60,61x, but the approach appears more applicable to monoclonal antibody purification than to human plasma fractionation. 3. Future trends Application of affinity chromatography in current industrial plasma fractionation schemes faces two issues. One is the selection and development of ligands tailor-designed for the specific capture of proteins w62x from complex plasma fractions; the other is the integration of these new purification approaches in the existing, heavily integrated and regulated plasma fractionation process already in place. Apart from the extraction of Factor VIII, Factor IX, and Antithrombin III, affinity chromatography is not used as the mainstream tool for extraction from relatively crude plasma fractions, but rather as a downstream polishing step of pre-purified fractions. This is linked to the inherent technical limitations of current affinity media Žrisks of leaching affecting several products, limited flow-rate, relatively high cost, poor resistance to cleaning and sanitizing conditions., and to the complexity of the human plasma feedstock. In addition, for plasma fractionators to change an established complex fractionation process would represent a significant regulatory burden Žprocess validation


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and clinical trials. that must be justified by solid benefits, such as improved safety, yield, productivity, or overall quality. There are, however, two new ways of developing ligands that may have important practical applications for the production of plasma proteins. Phage display technology w63,64x allows the synthesis of peptides or proteins, including antibodies w65x that are expressed on the tip of a modified bacteriophage, therefore allowing the selection and characterization of ligands binding a protein of interest in a plasma feedstock. This approach makes it possible to generate a vast library of peptides for finding compounds that bind to a targeted protein. Further selective refinements allow isolating ligands that present optimal performance with respect to binding and elution to fit industrial productivity and optimal yield Že.g. appropriate pH and ionic conditions for binding and elution, capacity, resistance to cleaning and sanitizing procedures.. Such ligands can then be produced in sufficient amount by recombinant technology or, if they are of small molecular weight Žsmall peptides., by chemical synthesis. The same in vitro approach can also be used to produce artificial antibodies w66x. Combinatorial chemistry is based on analysis, e.g. by crystallography, of the interaction between a protein and a known ligand. It leads to the synthesis of complex chemical structures. Results are exploited to re-create, piece-by-piece its contact surface and to study interactions with proteins. A combinatorial library for new affinity ligands exhibiting structural diversities can then be produced and evaluated. Both approaches, which can be used jointly, allow high throughput screening and have the advantage of being amenable to large-scale synthesis. In the field of plasma fractionation, they can overcome the current significant drawbacks of, e.g. murine antibody ligands that are expensive to produce and possibly unstable in harsh sanitizing procedures. They can also be used to replace other ligands derived from animal source Že.g. heparin or gelatin. that raise concerns about potential infections Že.g. prions, parvovirus.. Using such very specific ligands, product purification can be carried out, in principle, by simple filtration and elution, as non-specific binding should be minimized during the screening phase of the ligand. One can expect that peptide affinity chromatography may find application in the purification of important proteins like Factor VIII w67x or von Willebrand Factor w68x and others. Application to the removal of contaminants, including viruses, is also possible w69x. Availability of highly specific, tailor-made ligands opens the way to the development of a technology for capturing plasma proteins from crude plasma fractions. A ‘cascade capture’ approach would allow the extraction of fragile or trace plasma proteins, such as Factor VIII and other coagulation factors, directly from whole plasma, thus obviating the use of preliminary crude extraction steps Žsuch as cryo-precipitation. that suffer from low recovery and potential denaturing effects. New capture technology should significantly improve protein recovery, a crucial element, considering that human plasma is available in limited amount and that the world needs for several plasma products are far from being covered by the current supply. Considering the considerable progress and expectations afforded by new protein affinity purification approaches, it would seem logical that novel applications will be seen in the field of plasma fractionation in the future.

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