Displacement: Chromatographic Concentration ... - ACS Publications

Chapter 6

Displacement:

Chromatographic Concentration Control Jana Jacobson

Downloaded by UNIV MASSACHUSETTS AMHERST on August 2, 2012 | http://pubs.acs.org Publication Date: May 24, 1993 | doi: 10.1021/bk-1993-0529.ch006

BioWest Research, P.O. Box 135, Brisbane, CA 94005

High performance displacement chromatography (HPDC) is a practical, convenient means of controlling product concentration in protein purification to avoid extremes of concentration that can be deleterious to product quality. This study describes the purification of a complex mixture of closely related peptides by reversed phase chromatography in the HPDC mode. A major goal of the study was to compare the performance of displacement chromatography with that of gradient elution that has been the conventional mode of separating such mixtures. HPDC yielded a maximum product concentration lower than that obtained by gradient elution chromatography. The very high product concentrations that were observed in gradient elution separations can lead to aggregation and precipitation of a product. Such untoward effects, however, can be avoided by carrying out the chromatographic separation in the displacement mode. Hence, HPDC offered potential advantages in the ease of manufacturing for the high purity production of therapeutic compounds of interest to the pharmaceutical industry. After scarcely two decades biotechnology has come of age, and established itself as an integral part, now, of the pharmaceutical and medical industries. The recombinant D N A technology that began as a powerful laboratory tool for understanding protein and cellular function has spawned a major industry for the large scale production of protein and peptide drugs for human and animal therapy. B y many measures, such as sales figures, numbers of marketed and in-development products, variety of clinical indications targeted and stock market indicators, the biotechnology industry has matured and established itself as a significant commercial and technological player. This maturation has also necessitated a streamlining of manufacturing processes and development timelines to ensure profitability in widely differing markets and to thwart the growing competition for certain therapies. The growing health care cost burden and attendant price sensitivity for pharmaceuticals together with the relatively large dosage regimen of certain indications contribute an important element of cost-consciousness i n the development of manufacturing processes i n biotechnology. Competition for treatment of clinical indications with the greatest potential increases the need to rapidly develop manufacturing processes. Furthermore, regulatory considerations limit the

0097-6156/93/0529-0077$06.00/0 © 1993 American Chemical Society

In Chromatography in Biotechnology; Horváth, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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changes that can be made to a process, so the rapidly developed process should remain feasible as production demands increase with product launch and market expansion. These additional factors have pushed process design considerations in biotechnology farther and farther from the separation methods traditionally associated with biochemistry as practiced in the laboratory. Chromatography, in particular, has attained an outsized importance in purification processes in biotechnology, and the efficient implementation of chromatographic steps-in terms of low cost, short development time and robust operation-has received renewed engineering attention. Chromatography is a workhorse of the bioprocessing industry for reasons that include the ubiquity and the familiarity of the technique in biochemistry laboratories, the high efficiencies and capacities attainable in packed columns, the wide variety of selectivities afforded by the varieties of available stationary phases, and the relatively simple scale-up of results obtained i n the laboratory. The importance of chromatography to the industry underscores the need for readily-implemented, robust column operating modes, especially in light of the competitive factors described above. While relatively facile scale-up of separations is commonly cited as one of the advantages of process scale chromatography, in practice the operating modes adopted to optimize manufacturing processes can differ markedly from conventional—mainly analytical or micropreparative-approaches as practiced in the laboratory. These differences derive from the additional constraints on equipment capabilities, product specifications and control systems imposed at large scale. This study describes the operational advantages of the displacement mode of chromatography (1) that has been advocated as a means for enhancing the capacity of chromatographic systems without sacrificing the high resolution obtained at relatively low column loadings in conventional elution separations (2-5 ). Displacement chromatography differs from elution chromatography mainly by the use of a solution of a compound with strong affinity for the stationary phase to "displace" the components of interest from the column. In operational simplicity, displacement chromatography resembles step elution chromatography, since only step changes in mobile phase composition are involved. Fundamentally, however, the displacement mode differs from elution chromatography in that the displacer adsorbs more strongly to the column surface than the feed components, unlike the eluent strength modifiers typical of elution modes. Furthermore, the adsorption properties and concentration of the displacer control both the rate and concentration at which the purified feed components exit the column. In both gradient and step elution modes of chromatography the product emerges from the column in a relatively narrow band, or peak, whose volume and concentration are a complex function of flow rate, column efficiency, eluent strength, gradient steepness, component adsorption properties and feed loading. The concentration profile of the product exiting the column in displacement chromatography differs markedly from those obtained in elution purification. The product in displacement chromatography leaves the column in a rectangular band at a concentration fixed by the displacer concentration and the relative adsorption properties of the displacer and product (7). In this study the important advantages of these features of displacement chromatography compared to the elution modes are discussed. Experimental Chromatography Equipment. The H P L C instrument was assembled from two Waters M o d e l 6000A pumps (Millipore, Milford, M A U S A ) , a Waters 720 system controller, a Rheodyne M o d e l 7010 injector (Berkeley, C A U S A ) equipped with 20 μΐ, 2 m l and 5 m l loops and a Perkin-Elmer Model LC-75 spectrophotometer detector(Norwalk, C T U S A ) . Fractions were collected with a M o d e l LC-100 Haake Buchler fraction collector (Fort Lee, N J U S A ) . The same H P L C instrument was used for the analysis of the fractions except that injections were performed by a Waters Model 710B W I S P auto-injector. The column effluent was monitored at 280 nm, and


6. JACOBSON

Displacement: Chromatographic Concentration Control

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integration of chromatograms performed by the Nelson Analytical (Cupertino, C A U S A ) data collection system. Materials. Synthetic luteinizing hormone releasing hormone ( L H R H ) was kindly provided by Dr. Suresh Kalbag of Genentech (South San Francisco, C A U S A ) . Trifluoroacetic acid ( T F A ) and isopropanol (IPA) were supplied by J.T. Baker (Phillipsburg, N J U S A ) . Water was deionized and purified with a M i l l i - Q water system (Millipore) or was obtained from Black Mountain (San Carlos, C A U S A ) . The reversed phase chromatography experiments employed the RP1 D i s K i t supplied by B i o West Research (Brisbane, C A U S A ) consisting of a 4.6 χ 250 mm 5 μπι column, carrier, displacer, and régénérant. Procedures and Methods. Operating Steps for H P D C . After equilibrating the column with the carrier, the sample was loaded onto the column. The dual H P L C pump was programmed to generate a step gradient from the carrier to the displacer in 0.1 min, following loading of the sample. The column effluent was monitored at 280 nm, and fractions were collected over 1 or 2 minute intervals. Following the D C run, the column was regenerated by washing to remove the displacer prior to reequilibration with the carrier. Operating Steps for Gradient Elution. After equilibrating the column with the mobile phase, the feed was loaded onto the column in the appropriate volume. The dual pump unit was programmed to generate a linear gradient from the start conditions to the final conditions following loading of the sample. The column effluent was monitored at 280 nm, and fractions were collected over 2 minute intervals. Following the gradient run, the column was washed with the solution containing 60% I P A and 40% water with 0.1% T F A , prior to reequilibration with the starting eluent. Analysis of Fractions. 40 μΐ aliquots of collected fractions were analyzed by reversed phase H P L C using a linear gradient from 18% I P A , 0 . 1 % T F A to 42% I P A , 0.1% T F A in water over 15 minutes. The runs were at ambient temperature and a flow rate of 0.5 ml/min. The column effluent was monitored at 280 nm, and the data integrated by the Nelson Analytical system. Results and Discussion Reversed phase chromatography (RPC) has developed into the separation method of choice for a wide variety of pharmaceutical compounds, and especially for small organic molecules and peptides. R P C is still gaining in importance for purification of proteins, although in certain cases the acidic hydroorganic eluents commonly employed lead to denaturation and loss of activity of proteins. Nevertheless, for those compounds able to withstand the conditions of R P C , it is one of the highest resolution separation operations available. To further explore the advantages accruing from operating R P C separations in the displacement mode, H P D C was carried out on a crude peptide mixture arising from solid-phase synthesis of L H R H . The crude mixture was used in these experiments as obtained after cleavage from the resin and therefore contained failure sequences, partially deblocked components and other compounds commonly observed in unpurified synthetic mixtures. R P C is well-established as a tool for purification of peptides, including synthetic peptides, most commonly by gradient elution chromatography. Therefore, purification of L H R H by H P D C was compared with gradient elution purification in order to examine the advantages of the former and to establish a basis for developing systems for reversed phase separations by displacement chromatography. Gradient elution chromatography is the standard means of purifying synthetic peptides after cleavage from the resin, and can be expected to be an important means of purifying peptides on a large scale. The approach in preparative elution chromatography is similar to analytical H P L C of peptides, and so i n principle establishing the separation conditions is relatively straightforward. This is one of the main advantages of gradient



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elution H P L C over alternative means of operating chromatographic process equipment: the ubiquity and familiarity of the analytical technique fosters a high comfort level with gradient elution that is not apparent toward other modes of chromatography. Figure 1A shows an analytical chromatogram of the L H R H crude mixture. Figure I B shows a chromatogram of gradient elution purification of 40 mg of the crude L H R H mixture. Owing to the high concentration, the peak in this chromatogram is off scale and truncated. The preparative separation shown i n Figure I B was optimized by using a longer separation time to improve resolution relative to the conditions employed for analysis of the peptides. L H R H absorbs strongly at 280 nm, and closely eluting contaminants were not resolved at this loading so fractions were collected and analyzed by reversed phase H P L C . The gradient in eluent strength tends to sharpen the peak so that the product may exit the column at a higher concentration than that at which it was loaded onto the column. The conditions of displacement separation were established by relatively small scale experiments involving injection of sub-milligram amounts of the L H R H crude mixture and following it with the displacer solution. The retention time of the feed mixture, that exited the column as a narrow band, was noted. A lack of tailing of the feed mixture band indicated that the displacer effectively sharpened the rear of the displacement train. B y noting the retention time of the band, quantitative determinations of the adsorption behavior of the displacer on the reversed phase column could be made. Figure 2 A shows an example of such a scouting experiment, involving the injection of 0.1 mg of the L H R H mixture onto the column. The displacement separation of the L H R H mixture was scaled up to 4 mg and 40 mg feeds, as shown i n Figs. 2B and 2 C . Scale up in displacement chromatography yields a longer displacement train without resulting in an increase in product concentration in the effluent. Figure 3 shows histograms for the 40 mg elution (Figure I B ) and displacement (Figure 2C) separations. The relatively high concentration of the eluites in the elution compared to displacement separation is noteworthy. Figure 3 B shows that within the displacement train, early eluting components are well separated from the main component, L H R H . The separation between L H R H and later-eluting components is poorer, suggesting that Figure 3B may not represent a fully developed isotachic displacement train. Nevertheless, most of the fractions containing authentic L H R H are of sufficiently high purity to yield a purified L H R H with high yield. The relatively high product concentration in the effluent obtained when gradient elution is used can also effect the degree of aggregation of the molecule. The peak obtained by gradient elution has a maximum concentration that is determined by the chromatographic system and the feed loading, so that high capacity is associated with a high concentration in the product band. Essentially the concentration at the peak maximum is uncontrolled by the operator. Furthermore, since bandspreading and the effects of overloading the column result in peak broadening, the maximum concentration of the product can be many times that of the average concentration obtained after pooling the collected fractions. A t this maximum concentration the product may undergo aggregation or other concentration-related changes. Concentration-dependent aggregation may be irreversible and lead to a loss in activity of the product (6). Displacement chromatography offers additional advantages compared to gradient elution separations because the product concentration is uniform i n the column effluent, and can be controlled by the operating conditions to prevent the extreme concentrations encountered in overloaded gradient elution chromatography. While the peptide mixture examined here does not manifest untoward behavior related to aggregation, such behavior has been reported for proteins in reversed phase conditions (7) and may be exacerbated under high salt concentration conditions as employed in ion exchange chromatography.


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Displacement: Chromatographic Concentration ... - ACS Publications

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