Enzymes in Biomass Conversion - American Chemical Society

Chapter 13

Chromatography in Enzyme Isolation and Production 1

Ronald M . Spears

Downloaded by UNIV OF BATH on June 26, 2016 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0460.ch013

Pharmacia LKB Biotechnology, Inc., Piscataway, NJ 08854

Recent advances in the development of chromatographic media and equipment have made chromatography a viable choice for protein isolation and purification. Gels more stable to chemical (i.e., concentrated sodium hydroxide) and mechanical pressure allow more efficient separations with greater reproducibility, and increased column cycles using clean-in-place (CIP) procedures. This paper describes the application of different chromatographic methods (i.e., affinity, ion exchange, and gel filtration) to large-scale protein purification, along with the merits of various column designs presently available. The benefits of process development and production automation will also be reviewed.

Many methods have been developed to separate substances of interest either from natural sources or from synthetic or fermentative processes. One method that has been successful in more applications than any other is chromatography. Chromatography has been used to separate small substances such as salts, minerals, and organic compounds (e.g. drugs) to more complex biological molecules such as proteins and nucleic acids. Chromatography is presently carried out both in aqueous and organic solvents, using a wide variety of supports from paper to dextrans, and silica to agarose. Chromatography has a gained special place in the separation of proteins and other labile complex biological substances due especially to its typically gentle nature. These procedures can provide well-defined separations of similar polypeptides while yielding a high percentage of the original biological activity of the materials separated. Furthermore, chromatographic separation procedures can be utilized for both analytical purposes where milligram quantities are isolated, to production scale where tens to thousands of grams per year are separated. Due to the labile nature of most proteins, separations requiring biologically active products are almost always carried out in aqueous solvents. The best success has been using a stationary, hydrophilic, non-interactive base support matrix physically or chemically modified for specific applications with biological agents to be separated in the mobile phase. This allows predictable and reproducible chromatography with the separation mode selected for that matrix by its manufacturer (e.g., ion exchange, affinity, etc.). Current address: 1702 North Orchard, Chicago, Illinois 60614 0097-6156/91/0460-0169$06.00/0 © 1991 American Chemical Society

Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Chromatography of material such as proteins has occasionally been carried out in batch mode. Here the proteins are adsorbed onto a chromatographic medium by mixing the sample and the media in a common vessel prior to specific elution of the various adsorbed proteins. The mixture's components are subsequently eluted from the media by various types of elution schemes (e.g., salt and/or pH steps or gradients, specific affinity elution, etc.). Chromatography also has its own special equipment requirements that vary dependent on the type and scale of the procedure, and the efficiency level desired from the separation. This paper deals predominantly with chromatographic separations as they pertain to large-scale industrial processes and their special needs and considerations.


Chromatographic Techniques and Their Applications Successful application of chromatographic techniques relies on resolution, or the resolving power of the particular technique used. Resolution is defined by the relation of selectivity and efficiency of the chromatographic gel media (i). Selectivity is a function of the mode of separation of the gel (i.e., gel filtration, ion exchange, etc.) and efficiency is a function of the support matrix (i.e., particle shape, size distribution, mechanical stability, density of interactive chemical groups, etc.). Each of the various modes of chromatographic separation have unique advantages that dictate where and when in a purification process these techniques should be used. Initial Product Capture. Initial chromatographic steps involve product capture or concentration. The feed stream, coming from fermentation, cell culture or tissue extrac tion, is generally clarified using filtration or a bioprocessing agent. After clarification, the most versatile and commonly applied chromatographic procedure is ion exchange (IEX)(2) on a solid matrix. IEX's popularity is due to its ability to handle large volumes of feed in a short time using a relatively small amount of a gel medium. Selection of proper loading conditions can insure that the percentage of the desired product bound is as high as possible while minimizing unwanted by-products. Elution with salts using either stepwise or linear gradients is then used to fractionate the bound products. Effectively designed IEX procedures can give tremendous product enrichment during the first chromatographic step. Another practical, but currently less utilized, technique for use at initial stages is hydrophobic interaction chromatography (HIC). Also a concentrating technique, HIC requires high ionic strength to promote protein adsorption making it a good technique to follow a salt precipitation step, or an initial IEX concentrating step, where the salt concentration in the eluted product fraction is high. Proteins are then eluted (in HIC) by reduction in eluent ionic strength. Both IEX and HIC can be used in batch mode where the chromatographic media and feed are mixed, the mixture put in a vessel, and a simple one step elution is used to displace the product (and co-eluting contaminants). The advantages of batch adsorption are minimal equipment requirements, and its simplicity to perform. However, it is very cumbersome at large scales and requires much operator manipulation of the media and the product. Loading the feed on IEX or HIC solid phase media packed in a column provides an efficient process where loading and elution of the product occurs in a controlled environment where the gel is not handled during processing. Moreover, because less gel is required, more protein can be loaded on gel packed in a column than in free solution (3). Even though the equipment costs (columns, pumps, etc.) are higher than batch mode, this cost is quickly recovered from savings in buffer and gel media. Savings in operator time is also realized because these steps are easily automated. After the initial isolation or capture step, the product is in a more workable


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volume (and buffer) and is then ready for further fractionation. Depending on the requirements for the next step, the product buffer can be changed using a desalting or diafiltration step prior to application on the next chromatographic column. Product Resolution. The selection of the separation step or steps, and the sequence of those steps, that follow initial isolation require as much knowledge of the protein as possible. This will enhance the ability to apply techniques whose selectivity allow the most efficient, most economic, and best achievable resolution of the product. Combining chromatographic techniques in a logical sequence affords the most efficient process (see Fig. 1). Chromatographic media properties such as capacity, recovery, resolving power and selectivity should all be considered in establishing the sequence. There are two design rules commonly adhered to by process development chemists. First is to utilize higher capacity techniques such as IEX, HIC or affinity earlier in the scheme when higher volumes are commonly found. The second is to use different chromatographic techniques for each step to take advantage of the different selectivity types available. The most commonly used resolving techniques in a process are IEX and affinity (4). Both techniques lend themselves easily to scale-up and provide the greatest resolv ing power. HIC is gaining popularity, but is limited in both application and predictability. IEX is by far the most versatile of the resolving techniques as it can be applied to the greatest number of proteins under the greatest variety of conditions (2). Manipulation of buffer conditions such as pH, ionic strength and elution schemes (i.e., stepwise or linear gradient) affords tremendous potential in the ability to optimize IEX separations. An optimized IEX separation is relatively inexpensive to operate since simple salts are commonly used for product elution. In addition, since base-stable IEX gels are commercially available, cleaning and depyrogenation are effected with inexpensive agents such as sodium hydroxide or ethanol. From a regulatory standpoint, it is also very easy to document the removal of both the eluents and cleaning agents from IEX column effluents. Affinity chromatography, although not as versatile as IEX, offers the highest resolution available where the technique is applicable. Using a column filled with an immobilized ligand that is specific only to the product, a single pass of the product fraction over that column can often yield product pure enough to require only a final polishing step. Affinity chromatography is particularly attractive because of the simplicity of its operation. As this technique is most often carried out as an adsorption/desorption technique, it is no more complicated to operate than gel filtration. However, unlike gel filtration, very large feed volumes can be processed on a relatively small column in a very short time yielding small volumes of a relatively con centrated product. This allows unsophisticated, relatively inexpensive equipment and control systems to be used. The major drawbacks of affinity chromatography are the high cost of affinity gels and the labile nature of some affinity agents (e.g., proteins and antibodies). Also, the immobilized ligand is often sensitive requiring the user to clean and depyrogenate the gel with agents much more expensive than common cleaning agents such as sodium hydroxide. There is also the requirement to effect removal of eluent and trace amounts of column ligand from the product and the additional time and costs needed to validate those procedures. Nevertheless, the resolving power of affinity makes its use very compelling since the economy of operation and product purity achieved provides savings which can far outweigh the higher initial and operating costs of the procedure. Gel filtration is the last of the major chromatography techniques commonly applied in the resolving portion of a process. Of all the techniques discussed thus far in this chapter, gel filtration offers the lowest resolution. The separation is based solely on Stake's radius of the protein molecule and is the most sensitive to flow rate and sample volume. To achieve significant resolution among sample components, the sample volume should be no greater that five percent of the column bed volume.



Gel Filtration

Figure 1. Examples of logical sequences of chromatographic steps.

Affinity

Affinity

Ion Exchange

Gel Filtration

Affinity

HIC

Affinity

Gel Filtration

Ion Exchange

Hydrophobic Interaction

Ion Exchange

Ion Exchange

Gel Filtration

Hydrophobic Interaction

Ion Exchange

Ammonium Sulfate pptn

Ion Exchange

Ammonium Sulfate pptn


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Obviously, with large sample volumes, the column and gel requirements result in a costly process. It is for these reasons that gel filtration is selected as the last or among the later resolving steps in a process, where both the sample volumes smaller, and the mixture to be fractionated is far less complex. Less product manipulation between chromatographic procedures (i.e., buffer exchange, etc.) reduces total process time and increases overall product yield. This is accomplished by considering the compatibility of the targeted techniques. For instance, the need for buffer exchange, concentration or lyophilization of the product can be reduced by placing diluting techniques after concentrating ones (i.e., gel filtration after IEX or affinity). Physical handling of the product can be minimizedfartherincreasing the yield of the process. Other common examples include following IEX with HIC, or salt precipitation with HIC where the product fraction from the previous column can be directly applied to the second column with minimal preparation. Polishing. This last process step prepares the product for final formulation or for actual sale. It is designed to remove any aggregated protein, remove residual chromatographic eluent(s), and place the product into a specific solvent. These requirements are admirably served by gel filtration. At this point, the sample volume is small and the product fraction to be applied is fairly clean. The gel and column equipment requirements are now within reason and, the clean samples result in much longer gel life. IEX can also be used for polishing. Although usually too costly to be applied earlier in a process, high performance ion exchange chromatography (HPIEC) has been used to polish small quantities of high value proteins. HPIEC offers high efficiency which increases resolution resulting in high purity products. IEX has also been used for final concentration followed by a step elution of the product with a volatile buffer (e.g., ammonium bicarbonate) allowing lyophilization for storage or sale. Chromatographic Supports Chromatographic resolution is also dependent on column efficiency (1). Column efficiency is directly dependent on the nature of the support matrix and how well that support is packed in its column. Available chromatographic supports are based on dextran, agarose, polystyrene, acrylic, cellulose, silica gel and a variety of other polymers. Although cellulosic supports are manufactured in both microcrystalline and beaded forms, most supports are beaded. Newer supports may use hybrid bead construction where the base support is coated with a second material (e.g., dextran or silica coated with agarose). The most effective supports used in the separation of proteins all have certain common characteristics. They should be hydrophilic as separations are almost always carried out in aqueous buffers. Supports must be inert in that nonspecific binding is minimized. It is also desirable that the support does not contribute to the separation in ways different from the active groups attached to it. This helps to insure predictability and reproducibility of the separations among different manufactured lots of chromatographic media. The support should be stable to common cleaning and sanitizing agents such as sodium hydroxide. Cleaning allows the maximum number of separation cycles on any one column while preventing product contamination across product lots. Caustic cleaning also sanitizes the gel and helps remove pyrogens. Stability to mild organic solvents (e.g., alcohols, acetone or acetonitrile) is also desirable. These solvents can be used for removal of lipids and other hydrophobic contaminants from the gel, enhancing the solubility of certain feed stream components or simply for their bacteriostatic effect. Lastly, a good process support should have good mechanical properties. Many of the better cross-linked supports can handle linearflowrates in excess of 250 cm/hr without


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compression of the packed column bed and are also not subject to shrinking and swelling with changes in ionic strength. These characteristics allow all the chromatographic regeneration and cleaning procedures to be performed in place in the column thus eliminating the need to unpack the column every time the support needs to be cleaned or regenerated.


Chromatographic Equipment Careful selection of equipment used to carry out chromatography is as important as the selection of the technique itself. Flexibility, ease of manipulation and ability to automate are important. Different techniques have different requirements for column configuration, all require the maximum throughput for process efficiency and economy, and all benefit from automation of the individual process steps. In the case of IEX, HIC or affinity chromatography, the desired column configuration is one of short bed height and large cross sectional area (1). This will allow high throughputs with low pressure drops reducing the cost of both the column and the pumping and support systems. However, more importantly, high throughputs will reduce the process time reducing labor and utility costs. The added advantage of short bed heights is the ability to use softer support matrices with less fear of restriction of eluent flow resulting from overcompression of the gel bed. In contrast, gel filtration requires taller thinner columns (i), the height determined by the resolution required. Newer less compressible gels can be packed as high as 50 cm without any significant reduction in manufacturer's specified flow rate ranges (with gradual reductions corresponding to increased bed heights) (5). Older and softer more compressible gels require multiple columns to be placed in series to attain the same overall bed height (6). This configuration combines both the benefit of high throughput achieved with low bed heights and the resolution obtained with longer columns. The three basic column designs are presently commercially available (Fig. 2a-c). The first two are straight through tube designs and differ in the end piece construction. The first of these two usesfixedend pieces and holds a specified amount of gel. The amount of gel used in the packing slurry must be sufficient to fill the column as no end piece adjustment is possible. The second of the straight through designs utilizes an adjustable end piece. The obvious advantage is that the top column end piece (adaptor) can be adjusted to the top of the gel bed after packing compensating for any inaccuracies in calculating the amount of gel present in the packing slurry. However, columns of this design are more difficult to clean and sanitize as there are more dead spaces around the adaptor than in thefixed-columndesign. The third design is the radial flow column where flow moves from the outside of the cylinder to the inside. This provides high throughput and the ability to use soft gels. Unlike the straight through designs, it is difficult to pack this column efficiently. Hence, its best applications are affinity and simple IEX or HIC where the separation is a simple on and off procedure. The Basics of Scaling-Up Chromatographic Separations In this brief treatise the discussion of the scale-up of chromatographic processes can not be fully covered. I will attempt to summarize a few of the general rules that most process development chemists attempt to follow to make the transition from the bench to the pilot and production scale easier and more efficient. One primary goal is to conserve selectivity throughout scale-up. This means attempting to use the same separation chemistry in development that will eventually be used in production. This avoids the need to re-optimize in each stage of the scale-up process. It is desirable to use the same support matrix to avoid unforeseen matrix effects causing unnecessary modification of developed procedures.




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Figure 2a. Example of a typical fixed end-piece column.




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Figure 2b. Example of a typical adjustable end-piece column.


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Figure 2c. Cutaway diagram of a typical radial flow column. (Reproduced with permission from ref. 8. Copyright 1989 Astor Publishing Corporation.)



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Retention of linear flow rate is crucial to the scale up of any chromatographic technique. It is important to maintain the same ratio of the sample volume and the elu tion volume to the column bed volume, and to keep the sample concentration constant. Adherence to these to principles avoids variation in available dynamic capacity due to changes in the point of "sample breakthrough" (7). It also helps maintain the desired and expected peak shapes making design of process operation easier. Maintaining column dimensions that remain proportional throughout the scale-up process insures predictable results. Due to wall effects on both fluid flow through the column and support of the gel matrix, columns with a minimum diameter of 10 cm must be used in the initial steps of process development (wall effects are negligible at this diameter). Application of the diagnostic tools of HETP (height in equivalent theoretical plates) and peak symmetry throughout the scale up process will insure that the maximum performance of the selected gels is attained (1). Information from these diagnostic techniques can also serve as a standard for evaluating newly packed columns. HETP and peak symmetry measurements, when compared to a set standard, can also indicate an aging column and provide a basis for the establishment of a standard operating procedure in the production process. Literature Cited 1. Process Chromatography, A Practical Guide; Sofer, G. K.; Nystrom, L.-E., Eds.; Academic Press: San Diego, CA, 1989; App. A . 2. Scopes, R. Protein Purification Principles and Practice; Springer—Verlag: New York, NY, 1982. 3. Spears, R. M . Ph.D. Thesis, University of Maryland, College Park, M D , 1982 4. Bonnerjea, J.; Oh. S.; Hoare, M . ; Dunnill, P. Bio/Technology 1986, 4, 954-958. 5. Haff, L. Α.; Easterday, R. L. J. Liquid Chromatogr. 1978, 1. 6. Cooney, J. N . Bio/Technology 1984, 2, 41-43, 46-51, 54-55. 7. Christer-Jansson, J. Adv. Biochem. Eng. 1982, 25, 53. 8. Saxena et al. BioPharm 1989, 2(3), 48. RECEIVED December 6, 1990


Enzymes in Biomass Conversion - American Chemical Society

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