Downstream processing of proteins: Recent advances

Biatech. Adv. Vol 7. pp. 467-488, 1989 Printed in Great Britain. All Rights Reserved

so.oiJ + .50 1989 Pergamon Press plc

073P975W89

0

DOWNSTREAM PROCESSING OF PROTEINS: RECENT ADVANCES JOHN

R. OGEZ, JAMES C. HODGDON, MARC STUART E. BUILDER

Genentech. Inc., 460

Pt. San Bruno Blvd., South Son Fmncisco,

P. BEAL and

CA 94080, U.S.A.

This review on the downstream processing of proteins describes innovations that have occurred in the field since 1983. Several areas have seen particularly high levels of achievement, and are accorded expanded coverage relative to our previous review [l]. As an example, the increasing integration of downstream operations with upstream technologies, such as mokcular biology and fermentation, has led to the development of some very powerful processes. The degree to which organizations understand that there needs to be one unified process, rather than the independent steps of cloning, fermentation and recovery, seems directly related to the ultimate speed and success of the development effort. In 1983 one of the most active development areas was chromatography, especially affinity chromatography. This is still true today, and this topic has been expanded to include biospecific adsorptions that would not traditionally be classified as chromatography. with more proteins being developed for human administration, there has been an increased emphasis on all aspects of process h giene. In addition, there has been much discussion about the impact of regulatory deman J s on the design and development of the manuficturing processes. Therefore, a section has been added which covers several of the regulatory issues that have been raised for products of the new biotechnology. Finally, as some of the early process development achievements are now beginning to bear fruit in the form of patents, we have increased our citation of this area of the literature. INI’ERACITON OF DOWNTREAM PROCESSING WITH MOLECULAR BIOLOGY AND FERMENTATION Downstream processing of proteins, in the narrowest definition, is the purification of proteins from conditioned media or fermentation broths. However, many controllable fictors that influence purification occur early in the production process. It is the integration of downstream processing with upstream operations, such as molecular biology and fermentation, which provides some of the largest downstream opportunities. The choice of host has a dramatic effect on downstream processing. Depending on the protein and its intended use, one or more of several host systems mentioned below may be candidates for cost effective production. The choice depends on many factors. For instance, the fermentation and cell harvesting conditions differ substantially for a fusion protein produced in a bacterium compared to a protein secreted by a mammalian cell. As another example, the synthesis of glycoproteins of human origin in yeast, insect, or mammalian cells may result in cai-IxAydrate moieties that are different from each other and from the natural product [Z].

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Molecular Biology Since the inception ofgenetic engineering, E. co/i has been the principal host for the expression of recombinant proteins. Most proteins cloned into E. roli are expressed either with the native sequence (with methionine N-terminus) [3] or as fusion proteins with a II& crobial-derived leader sequence [4]. The soucture of the leader sequence in a fusion protein can be e loited to ease downstream processing, as exemplified by Moks et al. [r] who fused insulin- IX e growth factor (IGF) to the C-terminus of a Protein A-derived leader. The Protein A moiety confen affinity for the Fc region of antibody molecules, enabling the affini purification of the fusion rotein. The IGF is then cleaved from the leader using hydroxy73mine. In another approac g , Brewer and Sassenkld [6] fused polyarginine tails to the native se uence, causing it to have an unusually high isoelectric point and to bind tightly to cation exex ange resins. After digestion of the polyarginine with carboxypeptidase B, the product was then rechromatographed under the same conditions, and separated from the basic contaminants which bound to the resin. In some instances, the product is packa ed by the cell as a dense and insoluble “inclusion” or “refractile” body. In cases where me tk ods for protein refolding can be found, the density and composition of these particles can be exploited for the rapid initial purification of the polypeptide chain 171. More recently, reduction systems which direct secretion into the periplasmic space [8], or into the extracel Pular medium 19;IO], are becoming more widely used. There are several advantages to having the product transported into the periplasm or secreted outside the cell. Transport across the membrane is usually accompanied b cleavage of precursor forms to yield mature N-termini. In addition, the milieu outside t g e cytoplasm can be more conducive to formation of disulfide bridges. An added benefit is that cell disruption can be carried out under milder conditions for periplasmic secretion, and eliminated altogether for extracellular products. In any purification, it is advantageous to start with the highest possible ratio of product to contaminants. This is one reason many attempts have been made to use molecular bi010 to increase expression yields. For example, Shen [I l] has successful1 joined multiple ins 3 n genes as a means of preventing product degradation in E. coli. Go x et al. [I& 131 showed that cloning of a tissue plasminogen activator or human serum albumin gene into E. coliresulted in increased production of cell proteases and heat shock proteins that degrade or somehow lead to the incorrect processing of these cloned proteins. They reported that when cells manufacture a new protein encoded by a foreign gene, the cells often view it as foreign and implement defensive mechanisms that degrade the newly produced protein. Having identified this defensive mechanism, new strains of E. cob have been developed that are deficient in it ~141. For many proteins, eukaryotic expression systems are now preferred because of their ability to post-translationally modi polypeptides (e..d” by glycosylation or mrnacarboxylation), to properly ?old the poiypepa e chain and to form disulfide bonds. A Eaost that has been widely employed in the beer and wine industry, and is now being seriously ursued in biotechnology, is the yeast Succbaromyces cerevisiae[lS]. Calf chymosin II61and Peucocyte interferon ~171are two e:,:>mpies of proteins that have been successfi~lly produced in yeast. However, difficulties can aT:se when this species is used as a host. Wt L’IIhel?atitis B s&&e antigen is expr-ssed in yeast, a mixture of chemical forms COIII\JIi\i”l! ‘1)t . monomersand disulfide-bonded dimers is produced. The desired tlisull~tl~~al:{! e< ,*led UC.

DOWNSTREAMPROGESSINGOFPROTEINS must then be created by including a specific oxidation step in the recovery process [18]. Recombinant proteins have recently been expressed in insect cell lines through the use of a baculovints expression vector. The baculovirus system shares many of the advantages of mammalian systems and has the added advantage of being less fastidious. This system has been used recently for the production of recombinant human interleukin 2 119~and human alpha- and beta-interferons 120;2 11,as well as for the production of recombinant human immunodeficiency virus envelope glycoprotein (22; 23;241and soluble CD4 protein which is directed against HIV [zs]. The problem of the relatively low productivity of mammalian cells is being gradually overcome [26], asexemplified by the use of the dihydrofblate reductase selectable marker in Chinese hamster ovary cehs in order to increase the copy number of the desired gene (271. tine

Fermentation and CeR Culture One of the most important areas of interaction between downstream processing and fermentation is the development of suitable media for cell growth. For example, in mammalian cell culture the use of medii supplements such as serum may increase cell density and viability and, in mm, product concentration, but ma adversely affect the overall recoverability by leading to complex formation and product Begradation [28; 291. Another example is the determination of the time of harvest. Often, allowing a culture to run longer results in an in crease in titer, but with a concommitant increase in debris and degraded forms. Thus, the selection of the optimal harvest time must inchrde consideration of the effect on recovery operations. The method of krmentation can have substantial impact on the subsequent purification of protein. For example, the owth of cells by batch rather than by continuous (i.e., perfusion) culture can lead to time- l!rependent differences in the protein product or in the spectrum of contaminants and &ii in turn often affbcts behavior during downstream processing [30; 31;32;33;341. CELL SEPARATION The recovery portion of a process usually begins with the separation of cells and particles from the fermentation broth or conditioned cell culture medium. An excellent overview of the various solid-liquid separation processes and the considerations that should be followed when selecting a particular process is resented b Bowden [JS]. The two main ’ de choice of method depends options for cell separation are centrifugation and I!In-anon. primarily on whether the protein to be recovered is contained inside the cell or is secreted into the medium. The requirement to maintain containment of live recombinant cultures is an additional factor that can affect the choice and design of harvest method and equipment. In order to comply with government guidelines for the containment of recombinant organisms, the fermenter contents may be treated in some way to greatly reduce the number of live cells prior to harvest. For example, the broth might be heated to greater than 60” C or adjusted to a strongly acidic PH. Alternatively, the cell membrane may be disrupted by passage through a me&anical disintegrator or by treatment with detergents or organic SOL vents. Each tvoe of inactivation treatment can have a different effect on the efficiency of centrifugationbr filtration. The most important variable is the particle size distribut!on of the generated suspension. On the (11her hand, one may decide to perform the harvest on live cehs. III this case, the harvest equipment must be an integral part of the Con~JinlneIl~ system,

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and one must consider not only its performance under normal operating conditions, but also how well the system will work in the event of operator error or catastrophic equipment failure. Centrifugalion Bacterial cell separation is most often achieved by centrifugation beause of the solids content of these broths. Significant improvements continue to be made in several aspects of centrifuge design and construction. The use of stronger alloys has led to an increase in available gravitation forces of 80-90% in just the last few years. Other design improvements have been introduced to deal with containment and sanitization, which are two special concerns of recombinant pharmaceutical production [36]. Centrifugal separation ‘can be improved by conditioning the suspensions in order to increase particle size, reduce viscosity, and increase the density difference between particles and the medium before centrifbgation 137~.Pretreatment ma include flocculation of the bacterial cells by neutralizin their negative surface charges x rough the addition of di- or poly-valent metal ions or po Pyelectrolytes. The resulting sedimentation rates can be as much as 2000 times greater than that of untreated broths. Flocculation is especially important when centrifuging homo enates where the particles are very small. It must be noted, f owever, that tight controls are required to consistently reap the full benefits of induced flocculation. Floes are generally sensitive to shear and may easily break up in pumps and cenaifuge inlets. Successful flocculation is a balance between the reduction of sufficiently stable floes (e.g., by the use of strongly cationic polyelectrol es or 1y mechanical treatment) and minimizing co-precipitation or damage to sensitive pr IXLtS such as proteins. In some cases it is advantageous to divide the primary separation into two steps: one thickening step using a decanter centrifuge for concentration of tbe cells and one higher-speed centrifugal separation step for final clarification and total recovery. Filtration Filtration is a frequently used alternative to centrifugation. Zahka and Leahy 1381 claim that the principal benefit of cross flow filtration relative to conventional centrifugation is more efficient se ration because filtration systems can yield sterile filtrates, and such systems may be cons1*8” ered to be contained harvest devices. In addition, filtration rates are independent of density differences between cells and the medium. Another advantage of filtration is that cell or particle washing by diafiltration can be carried out in tandem with the harvest operation. Tutun’ian [39] reviews the two basic configurations for cross-flow membrane filtmtion, hollow ti ber and flat sheet, and weighs the pros and cons of each. The principal advantages of the flat sheet systems over hollow fiber cartridges are that they can be operated at much higher transmembrane pressures, and can be operated either with (for bacteria and east) or without (for mammalian cells and organelles) turbulence romoters. In comparing Kouow fiber filtration to centrifugation for harvesting bacterial an B yeast cells, he states that even with low flux rates, the economic advantages of filtration can be substantial, particularly when dealing with smaller cells or particles, where centrifuge throughput drops significantly.

DOWNSTREAM PROCESSING OF PROTEINS

CELL DISINTEGRATION Mechanical disintegration is the most common form of cell disruption used in industry today, although treatment with acids, bases, detergents and solvents has been found to be effective. The use of mechanical disintegration for large scale disruption of microorganisms has recently been studied by Schutte et al. [40;411, who examined two types of mechanical disinteklrtors: the LME ZO-mill and the Gaulin type M3-WI’BS. They calculated that for yeasts the capacity of both units is SO-70 kg/hr. However, for bacteria, the Gaulin was superior, having a capacity of SO-100 kg/hr, compared to lo-20 kg/hr for the bead mill. They also point out that one must consider the heat generated by these devices. For example, during disruption of yeast cells in a Gaulm press, the adiabatic rise in temperature of a 40% (w/v) suspension was linear, 2.2-2.4”C per 100 bar, over the pressure range tested (100-550 bar). Other pressure homogenizers similar in principal to the Gaulin are the Fren&-Press and the X-Press, which can be used on either frozen or liquid suspensions. Edebo [42] describes in detail the use of the X-Press, citing numerous references of its application in the disintegration of bacteria, yeasts and fungi. PRECIIVTATION

AND SOLID-LIQUID

SEPARATION

Fractional precipitation of proteins is a purification method which dates back to the last century, and it is still a useful technique for inexpensive and very large scale fracrionation. While it usually is limited by low resolution, it can be a helpful first step for concentration as well as partial purification. Hoare and Dunnill (431and Chan et al. 1441examined the kinetics of preci itation of food proteins using a variety of acids, salts, and organic solvena. They devise B a small tubular reactor that was useful for testing a large variety of precipitants on small s?mples, and set up a system for rapidly determining the quantity, stability, and rate of furmation of the precipitates. To date, centrifugation has been the most extensively used method for recovering protein precipitates since they are typically large, dense floes that have high sedimentation rates. In addition, the sticky, gelatinous nature of the recipitates often contributes to the rapid fouling of membrane filters. Dunnill and cowor 1:ers report that separation and recovery of protein precipitates by centrifu ation can be improved throu h better reactor design [45] and the acoustic conditioning o f protein precipitates [46], and ! ave proposed a process along these lines [47]. Devereux et al [48] have also compared ultrafiltration to centrifugation for recovering protein precipitates and have concluded that given proper equipment design and operating parameters, ultrafiltration can be an effective alternative.

FILTRATION

OF SOLUBLE

PROTEINS

Filtration, as a unit operation, continues to be used more fre uently in biotechnology and now finds applications ranging from the concentration and diafi9 tration of protein solutions to the perfusion culture of mammalian cells ~49;SO;~1; 52; 531. Considerations for scaling up membrane processes are reviewed by Tumnjian [s+. Techniques for cleaning and decontaminating UF membrane rearms with sodium hydroxide, sodium hypochlorite, phosphoric acid or other suitable chemir~l agents are reported b Ricketts er al. 1~51.Several useful articles dealing with general principals and applications o fyultratiltra~ioll ‘LO\! alsorecendy appeared in the literature [56; 57; 581.

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Experiments designed to find solutions to the problem of fouling of ultrafiltration membranes have been carried out by the lab of Reihanier and coworkers [59]. They doubt both the osmotic ressure and the gel-polarization hypotheses of membrane fouling, and indicate that a simp Pe particle filtration model involving the build-up of a protein cake of constant hydraulic permeability could adequately explain the observations. They also found that the build-up of protein on the membrane surtsce can be caused in art by the hydrophobic nature of the membrane itself. The initial build-up is dictated by II e interaction between the protein and the membrane wherein portions of adsorbed molecules adjacent to pores in the membrane effectively limit the flux. Further build-up of protein on the membrane can then occur b a secondary adsorption to the initially bound layer, leading to gel formation and further x w resistance. They propose the use of more hydrophilic membranes to significandy reduce problems of merfibrane fouling associated with ultrafiltration of solutions. The drawback, however, to the use of more hydro hilic membranes based on cellulose is that the tend to be less stable than their more hy Brophobic counterparts. Cleaning of hydrophilic ce rlulose membranes with extremes ofpH (less than pH 3.5 and greater than 10.0) or treatment with sodium hypochlorite should be avoided. Instead, detergents, weak acids or bases, or weak oxidants such as hydrogen peroxide can be used to re-establish initial flux rates while preserving the integrity of the membrane. AlEtlity ultr<ration The technique of affinity ultrafiltration, employing derivatized membranes, has recently be n to receive attention as an alternative to traditional affinity chromatography. Brandt et a $. [a01point out that a microporous membrane represents an “ideal” column geometry since it has an extremely short bed height (often less than a millimeter) and a large cross sectional area that is easily increased. Also, because of the inherent structural properties of UF membranes, the diffusion path to the ligand is ve 7 short, and.tfie efficiency of mass transfer is therefore ve high. Consequently, the extent o hgand partlclpanon remains high, even at very fast x ux rates. This becomes a particular1 im ortant considention when the ligand is relatively expensive, as for a monoc rona Pantibody or receptor. The fast mass transfer kinetics may permit a time-averaged compensation for the relatively lower volumetric capacity of membranes compared to chromatogra hit media. The relative value, advantages and disadvantages of affinity filtration compare B to affinity chromatography are still being actively debated. CHROMATOGRAPHY Chromatography constitutes the most diverse and powerful group of procedures for protein purification. Literally hundreds of media are available, and the uses to which they are put are even more numerous. A practical guide for im lementing chromatography operations in lar e scale processes has recently been published ! y Sofer and Nysnom WI. The following 8!lscussion on chromatography has been organized according to the major types of separations, such as size, charge and biological function. Ge.l Filtration Gel filtration, also called size exclusion chromatography, is one of the cornerstones of modern rotein purificadon. Even with major advancements in separatiorl t~‘~~llll’~,~ thal m-e

B over &e last three decades, the fundamentals of gel filtration rclrl IIII 1 t’:’ Iland.

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PROCESSING OF PROTEINS

Of the few pers specifically on this technique that have a peared in the recent literature, most have fEu sed on the optimization and maintenance o Plarge scale gel filtration systems. Drawing on Pharmacia’s experience in the plasma fractionation industry, Curling et al. [62] discuss the main fictors involved in successful and economic process chromatography. Using the human albumin process as an example, they point out the requirements for water, buffers, sanitization, and control. They make a strong case for using resins &at are base-stable since sodium hydroxide is cheap, effective, and easily removed. Ion FXchange Ion exchange is the most frequently used form of chromatography. Thii is due to its utility in initial concentration and purification and to the high capacity and economy of these resins. Notable recent trends in ion exchange include the increasing availability of hydrophilic, rigid supports and the increasing use of chromatofocusing, a powerful version of ion exchange chromatography. Recent applications of chromatofocusing include the separation of enzymes from tobacco lams [63; 641 and the isolation of CAMP-F from Streptucocc~ ugakuriac [65]. Scott et a P. [66] describe a chromatofocusing technique that substitutes a pH step-clution in place of the ampholytes, thus eliminating the necessity of later separating the ampholytes from the purified protein. This is important not only because it significantly reduces the purification cost, but also because it eliminates the regulatory issues associated with quandtation of residual ampholytes in the product. Nospecific Adsorption The stringent purity criteria imposed on protein therapeutics intended for human use have contributed to the widespread adoption of affinity chromatography as a pre arative purifiadon technique. Equations that an be used to predict the performance o P alXnity columns based upon such variables as the equilibrium and mass transfer constants are derived by Arnold et al. [67]. They describe how to use these equations and analyses to arty out the scale-up of affinity chromatogra hy [I%]. Additionally, they experiment with pulse techniques to characterize several a fi?*mty media [69]. Two reviews b Jan-Christer Janson [TO;711describe several large-scale appliations of affinity chromatograp g y, pointing out that in most cases, “true” chromatography never takes place. Whereas chromatography involves the simultaneous and differential migration of several molecular species, what takes place in affini chromatography is usually an all-or-nothing adsorption and desorption. Janson a7 so addresses several aspects of scale-up and describes a varie of configurations for carryin out biospecific adsorption, including packed columns, flu12 zed beds, and aqueous two- *p&se partitioning systems. Metal-&elate chromatography, or immobt tzed ion afiinity chromatography, is an emergin group-specific affinity technique. Porath and Belew [72] exphtin the under@ theory, d escribe the synthesis and suucmre of several resins, and discuss some of the ear fy results that were achieved. Sulkowski [73]reviews several model studies which led to an understanding of which protein strucmral features are primarily responsible for binding to metal chelate resins. Histidine and uypto han appear to be the residues of primary importance for binding. The presence of a sing Pe hi&dine residue on a protein’s surface is sufficient to allow binding to copper gels, and additional histidine residues increase the affinity. However, at least two proximal histidine residues are necessary for binding to iron gels. Several tryptophans are required to confer good binding to copper gels. Both papers point

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out the versatility and selectivity that can be achieved by the choice of the metal and suggest guidelines for the use of metal-chelate affinity chromatography. Reactive triazine dyes for large-scale affinity chromatography are attractive because they are cheap, can be covalently bound to resins in a single step, and exhibit high affinities for some proteins. The reviews b Janson [71]and Qadri 1741cover several large-scale applications of dye chromatograp g y, and also give a perspective of the historical exploitation of dye-protein interactions. For example, human albumin has been recovered from Cohn Fraction IV by using immobilized Cibacron BlueF3Gk The dye column, which had a 16 L volume, had a capacity of 300 grams of albumin per 6 hour cycle. One drawback to the use of dye resins for pharmaceutical production is the concern over potential toxicity of residual dye which may leak off the resin and contaminate the final product. This can be minimized by proper washing during media synthesis in order to extract all non-covalently attached dye. Producers of high-value therapeutics are turning increasingly toward immobilized monoclonal antibodies, because methods have been developed that permit reusing the media hundreds of times and because advances in hybridoma growth and antibody recovery have made them more economical. It is interesting to note that monoclonals themselves are often purified by affinity chromatography, for example, on immobilized Protein A. An extensive review of monoclonal antibody immobilization chemistry and use is reported by Chase 175; [761. When affinity chromatography is used on a commercial scale, one must consider column eometry and resin type. The economic demands of process-scale operations require that tBe throughput be maximized by using the highest possible flow rate (i.e., minimum residence time). As with other types of adsorptive chromatographies the maximum rate is determined by either gel compressibility or diffusion kinetics. In practice, the flow rate in large columns is usually limited by the compressibility of the gel, well before the diffusion limit is approached. Therefore, scaling up a packed bed is best accomplished by increasing the column diameter while minimizing the increase in bed height. A significant trend in affinity chromatograph is toward the use of more rigid sup arts, such as cross-linked agarose, dextran-coate dr..sd~ca, or hydrophilic-modified viny Palcohols and acrylates which permit l%ter flow rates. Two novel applications of affinity purification that use continuous extraction techniques have recently been devised. In the continuous affini recycling extraction technique of Pungor et al. 1771,the chromate aphy column is replace ? with two reaction vessels that contain the adsorbent. Sample is Ped into one vessel where it contacts the adsorbent. The desired product binds selectively while contaminants are diluted with the addition of wash buffer. The adsorbent with the bound product is then pumped into the other vessel where the addition of the desorbing solvent causes the product to detach. The regenerated adsorbent is recycled back to the adsorption vessel while the product is removed continuously through a membrane filter. A tandem continuous affinity purification/ultrafiltration technique has been reported by Luong et al. 1781for the purification of trypsin from pig pancreas in which the ligand is covalently attached to a soluble polymer. The soluble polymer and the eluting molecule are recaptured for reuse, thus increasing the economy of operation. HPLC Preparative-scale protein srp:lrarion by I-IPLC has grown rapidly OCR *IIPpast few years with developments in ion-exchange, reverse phase, hydrophobic interaction.

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PROCESSING OF PROTEINS

immunoaffinity, and size exclusion chromatography [79]. There are a number of ractical differences between analytical scale and process scale HPLC. Whereas analytica Pseparations emphasize maximum achievable resolution, preferably with the highest possible sensitivity, process HPLC attempts to separate the lar est possible quantity of a compound with resolution that achieves the desired level o f purity for the isolated substance. Thus, operational parameters for process HPLC are considerably different from those used for analytical application [EO].The amount of product loaded per m of column packing can be orders of magnitude higher. The HETP (height equivalent o $ theoretical plate) of process columns is usually greater, and the column is often operand at or near the overload regime. The equipment should be designed to be capable of continuous operation in a manuf&turing environment, reliable, simple to control, and easily serviced and maintained. It also must be designed to meet industrial safety standards for hi h pressure operation, electrical hazard, and explosion- roofing. The chromatography co Bumns and packings must have chemical and mechanica P stability that permit hundreds or thousands of injections, and column replacement must be simple and rapid [Sl]. Several manufacturers now offer systems that saiisfy these criteria, including Miliipore’s Kiloprep, Amicon’s K-Prime series; and the Protein Preo LC svstem from TosoHaas. The scale-un of ueotide and antibiotic muification by HPLC &ring s&h systems has been reported by Buigoynekt al. [SZ] and Dwye; [a]. FPLC is the trademark for Pharmacia’s medium pressure “fist protein liquid chromatography” system. The control and hydraulic mechanisms have in common with HPLC systems the programmability and precise solvent metering [84; 851. Al] materials that come in contact with the sample or solvents are inert and corrosion resistant. The system is supported with a wide range of hydro hilic chromatomedia that were specifically designed for separation of biopolymers (861. d e availability of these resins, which give high-resolution separations and high roduct recovery at fairly low pressure, is what distinguishes this system from conventiona ipHPLC equipment. The concept of the FPLC system has recently been scaled up in the form of the BioPilot system, which has sufficient capacity to be used as production equipment for some products. The design similarities between the FPLC and BioPilot svstems are such that. once an analvtical seoaration has been outimixed. it should be easily adaptable to process scale. Use of FPLC fo;monitoring prod&ion of ’ antibody to N&&a gunowboeaehas been reported [87], along with scale-up procedures for the purification of low molecular weight proteins from urine 1881. AQUEOUS

TWO-PHASE

EKTRACI’ION

Extensive reviews of aqueous two-phase systems are presented by Kula et al. [89; 901 and Mat&son [%I. This method has been used for separations ranging from the removal of cells and cell debris from protein solutions, to purification of one protein, such as interferon, from other proteins. In the technique of affinity partitioning, specific polymeric adsorbants arc used to alter the protein distribution coefficients, thereby greatly increasing the efficiency of two-phase extraction. Recently, a detailed economic analysis outlining the use of two-phase systems compared to other extraction and purification methods was done by Kroner et al. [92]. They claim the advantages of using a liquid-liquid s stem, namely high recovery, ease ofscalability, and continuous processing, outweigh the l!.igher chemical costs. Recycling of reagents (e.g., polyethylene glycol and salts) makes this method more cost effective at large scale.

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CTECHNIQUES

The electrophoretic separation of roteins is still predominately used as an analytical technique. However, Gobie et al. [WI an a Ivory et al. [w] have proposed the design of a device for preparative scale high resolution free flow electrophoresis. Electrodialysis has long been used at very large scale for the production of salt, potable water, and desalted whey protein, and is emerging as a useful unit operation in biotechnology applications. It allows desalting and buffer exchange of charged species, and the concentration of roteins in hi h yields. The fundamentals and applications of elecaodialysis have recently g een reviewe J by Reed [%I. PROTEIN

REFOLDING

Purification of active recombinant protein from bacterial sources, especially E. cd, often requires a folding or refolding ste The patents by Builder and Ogez [7; 961 and Rausch 1971deal with methods for refol ! mg recombinant proteins that are packaged into insoluble “refractile bAies.” In general, they suggest the use of strong denaturants such as guanidinium chloride or SDS to solubilize the denatured rotein under reducing conditions, followed b buffer exchange into a weaker denaturant sue ! as urea. Further purification is then usua Yy required, and the pure polypeptide is then fully refolded by gradual removal of denamrant. It is our experience that refolding yields are often higher when carried out with more pure preparations, since the presence of contaminants (e.g., components of the cell wall) can provide routes to improper conformations. Intermolecular aggregation during the refolding step may be further reduced by using a pH that is not close to the isoelectric oint of the protein . An alternative approach, in which the rotein is immobilized on a sol1-B support during the removal of denaturant, is reported ! y Light . [98]. For example, the fully reduced protein is bound to an ion exchange resin in the presence of urea, and the reductant and denaturant are removed by washing the column with a simple gradient of decreasing concentration. The immobilization prevents the formation of intermolecular disulfide bonds, and the kinetics of buffer exchange are easily controlled by the gradient shape and flow rate. Approaching the problem from yet another angle, Pigiet ans Schuster [w] report that a combination of thioredoxin and dithiothreitol has been used to reshuffle mispaired disulfides in bovine pancreatic ribonuclease A. In addition, an E. co/i-derived thioredoxin reductase system can be used to refold proteins containing incorrect disulfide crosslinks [IOO]. PROCESS

DESIGN

Careful design of the purification scheme and the subsequent optimization are essential to the success of a process. The cost of obtaining the product at the desired purity and in the necessary quantity depends principally on the choice and sequence of steps. Process optimization is another crucial element of putting a recovery process in place because it has a pronounced effect upon overall production cost and success rate. For example, in optimizing chromatographic operations, it is advisable to both clarify the feedstocks and develop regeneration procedures that allow reuse of the packing. This reduces hot11 downtime and labor required for column repacking and significantly lowers raw material and uality control costs. Man of the ;3m:: principles apply to the optimization CroSS-flow , Including regeneration procedures that allow membmne reuse Xltration operations as we1Y.

of

DOWNSTREAh4 PROCESSING OF PROTEINS

and determination of operating ranges of pressure and retentate/permeate flow rates that maximize flux. Sofer and B&ton 11011review the principles for incorporating chromatographic steps into protein purification schemes. They recommend using a hi h capacity technique, such as ion exchange, as an initial chromatographic step in order to ficr*‘iirate later purification steps by reducing process volumes. They also recommend using a highly s ‘tic technique, such as affinity chromatography, at an early point in the recovery, so that tR”e product is quickly separated from contaminants which would otherwise degrade it over time. They also suggest ways of linking multiple chromatogra hit operations in order to eliminam intervening buffer exchange steps, and address some of tI e considerations for scaling up column chromatography. Bonnerjea et a1 [102], surveyed 100 protein purifications that were published during 1984 and observed several clear preferences regarding the usage and ordering of various purification methods. For example, homogenization and precipitation were the most common initial steps, followed by ion exchange to partially purify and to reduce process volumes. Affini chromatography was generally used as the penultimate or final recovery step. In 45% of x e cases, the use of affinity chromatography resulted in essentially homogeneous product, regardless of the intial purity. Interestingly, neither aqueous extraction nor uln-afihration were used by any of the authors, perhaps because these operations can be conveniently replaced by centrifugation and dialysis at the laboratory scale. Hygiene For production of food and pharmaceutical grade proteins, hygiene is a critical part of downstream operations. Cleanin , sanitixation or sterilization of equipment is essential to avoid product contamination an d degradation that result from microbial growth. Several methods for maintaining chromatographic columns, particulatly antibody columns, have been described by Hill and Hirtenstein [ION]. The most important principles are operation in the cold and use of preservatives to inhibit microbial growth, and the use of sterilizing filters on column inlets. REGULATORY

CONSIDERATIONS

Characterization and Analysis Validation of processes to produce therapeutic proteins includes characterization and analysis of the host cell, the product of interest and impurities from the host cultute [ION]. Accordin to Petricciani [IOS],the characterization of mammalian cell lines used to produce biologr caI3products should include the history and genealogy of the cell lines, along with ot presence of adventitious agents such as mycokaryotyping, and testing for tumorigenici plasma, bacteria, fungi and viruses. The e?:rmination of teal or potential viruses from the final product deserves special attention, especially when mammalian cell lines or their products: (such as antibodies) are involved in the production process, as outlined by the World Health Organization 121.Martin [106] lists heat, pH extremes, chaouopes, detergents, and radiation as ossible means of viral inactivation, and gives examples of viral removal steps along with I eoretial calculations. For instance, with the Rous sarcoma virus ot murine leukemia virus, dropping the pH to about 3.5 for only a few moments provides 3 to 4 logs of inactivation, and heating to 56’C will generally give greater than 4 logs of killing within 5 IllillUtes.

477

478

J. R. OGEZ et al.

Nucleic acids represent another possible contaminant that must be quantified. Based on early concerns about potential oncogenic sequences, Petricciani [107]initially recommended that the final DNA content should not exceed “tens of picograms” per dose. Subsequently, however, experiments such as those done by Levinson et al. [108]demonstrated that as much as 10’ times more DNA (approximately 100 pgs) fails to initiate a tumorigenic response in immunosuppressed animals. These results have helped to reduce concern surrounding this safety issue. Proving that the purification process removes DNA to the picogram level often requires a combination using both in-process testing and validation of specific DNAremoval steps. , validation of DNA removal is achieved by spiking radioactive DNA into the starting monitoring its removal through the purification process. In cases where the dose is very small, it may be sufficient to assay the final product directly using hybridization techniques such as a “dot blot” assay. Examples of removal of spiked DNA by the purification process are reported by Obijeski et al. [lo91 in the purification of hepatitis B surface antigen, and Finter et al. [I IO]for the isolation of human lymphoblastoid interferon. A “dot blot” analysis of Roferon-A, in which 3*P-radiolabeled plasmid DNA is hybridized onto a nitrocellulose membrane filter, is reported by Bogdansky et al. [ill]. Residual cellular proteins must be quantified and, in some cases, characterized individually. The charact,:rization of protein contaminants in a recombinant human growth hormone preparation has been reported by Jones and O’Conner [112]. By growing the host, which lacked the growth hormone gene, in the presence of radiolabeled amino acids and then carrying out the normal growth hormone recovery process, they could obtain, quantify and identify the host-derived impurities. They also report an E. cofi protein assay that is sensitive in the 20 ngs/ml range. Petricciani [113; 114; I IS; 116)summarizes the recommendations for final product analyses.

Quality Control of Materials It is usually required that all e uipment and rea ems that come in contact with the product be subject to appropriate qua9.ny control chec & . All materials used in the manufacture of pharmaceuticals should pass quality control checks that include physical, chemical, and functional tests [117].

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