Large-scale process development for hydrophobic interaction chromatography. II: Controlling process variation (1995)

The scale-up of hydrophobic interaction chromatography (HIC) relies on users successfully anticipating the nature and magnitude of variations that affect the final process. Part 2 of this 4-part article examines both direct and indirect sources of variation in HIC processes and suggests ways to control them. These concepts can be used to improve scale-up and on-line manufacturing performance of HIC and other chromatographic methods. Introduction Scale-up failures, or failures of on-line manufacturing processes, are occasionally caused by external circumstances such as mechanical or electrical failure. However, most problems result from uncharacterized variations in process chemistry, raw materials, equipment, and the manufacturing environment. These problems are generally predictable and controllable. Controlling variation begins with identifying its underlying causes. Process developers can then assess the magnitude of variation. Some sources can be eliminated; others can be controlled. Remaining variations can usually be accommodated by altering process specifications. Adopting this proactive strategy significantly enhances scale-up success and helps to ensure dependability and reproducibility of online manufacturing processes. In addition to supporting good process economics, this strategy provides an invaluable foundation for process validation. Materials and Methods As noted in Part 1, we obtained Source 15ETH, 15ISO, and 15PHE hydrophobic interaction prepacked columns and bulk media from Pharmacia Biotech AB (Uppsala, Sweden) (1). All three media are based on 15-mm dp monodisperse spheres with a pore-size distribution suitable for large proteins. The base matrices are composed of poly(styrene–divinylbenzene) coated with a hydrophilic polymer. We obtained different monoclonal antibodies from Becton Dickinson Immunocytometry Systems (San Jose, CA) for this study and used them as process models. We purchased buffers and salts from Sigma Chemical Company (St. Louis, MO). All buffer components were American Chemical Society (ACS) grade or better. Process water was prepared using reverse osmosis and deionization. We filtered buffers through a 0.22-mm filter immediately after formulation and assigned five-day expirations. The experimental methods are described in the figure legends. Results and Discussion Process-related variations. The most important factors influencing the robustness of HIC scaleup are gel selection, binding conditions, and sample-application methods. These issues were discussed in Part 1 of this article (1). Temperature is another process-related source of variation. For most proteins, retention on HIC media increases with temperature (2–4). This appears to be a compound effect, resulting both from protein conformational changes and from modification of the degree to which salts alter water structure (2,5,6). As a result, HIC processes are benerally more sensitive to temLarge Scale Process Development for Hydrophobic Interaction Chromatography, Part 2: Controlling Process Variation Pete Gagnon,1 Eric Grund,2 and Torgny Lindback 2 This article originally appeared in BioPharm 8(4) 36–41 (1995) and is adapted with permission. Reprints of this and other BioPharm articles can be ordered through the Advanstar Reprint Office by calling Mary Clark at (541) 984–5226 1Validated Biosystems, Inc., 5800 North Kolb Road, Suite 5127, Tucson, AZ USA 85750 2Pharmacia Biotech AB, S-751 82 Uppsala, Sweden perature variations than other methods. Figure 1 shows that maintaining the selectivity of an antibody fractionation across a temperature reduction from 23 °C to 4 °C required increasing the ammonium sulfate concentration by approximately 25%. Other proteins may be affected to greater or lesser extents and should be evaluated on a case-by-case basis. The most common cause of temperaturerelated variations in HIC processes is insufficient equilibration of the sample. When using samples as small as a few microliters during early process development, even cold samples will equilibrate rapidly enough that temperature effects are negligible. However, as process development proceeds into evaluations of capacity and other parameters that require larger sample volumes, temperature effects are amplified in parallel. At full process scale, a too-frequent cause of process failure occurs when sample is taken directly from cold storage for a room-temperature process (Figure 2). Failures of this sort pose the worst obstacle to subsequent investigations because the evidence is lost as soon as the sample temperature reaches equilibrium. Avoiding this type of problem is largely a matter of education for both process developers and manufacturing staff. It is important that temperature specifications be included in manufacturing standard operating procedures (SOPs). SOPs should emphasize that all process materials—buffers and raw product— must be at a specified temperature before beginning a process. Minor temperature variations between development and manufacturing laboratories are unlikely to cause serious process deviations, but they should not be dismissed. Consult facility records to determine the range and average temperatures of manufacturing areas—then develop processes to those specifications. Variations from raw materials. Raw materials for manufacturing include raw product, buffer components, process buffers, and gel media. Routine variations in these materials seldom result in process failure, but they do have an impact, so it is important to characterize and accommodate them. Raw product. Typically, the raw product is the least controlled component in a purification process. Even under the best circumstances, significant variations in product concentration and the proportion of contaminants are likely. Very often, users conduct early process development with materials that are inconsistent with materials to be used in the final production process. The worst possible situation is when process developers use a failed production lot—such as one with micro2 Figure 1. Maintaining selectivity by increasing the binding salt concentration after reducing temperature from (a) 23 °C to (b) 4°C. Column: 30 mm x 6.4 mm Source 15ISO; buffer A (a): 1.55 M ammonium sulfate, 0.1 M sodium phosphate (pH 7.0); buffer A (b): 1.95 M ammonium sulfate, 0.1 M sodium phosphate (pH 7.0); buffer B: 0.1 M sodium phosphate (pH 7.0); flow rate: 940 cm/hour (5 mL/min); sample: 1 mL mouse IgG1 ascites. Conditions: equilibrate column with 5 mL (5 column volumes) 80% A; load sample by on-line dilution, 20% sample, 80% A; wash with 2 mL 100% A; elute antibody with a 10-mL linear gradient to 100% B; strip column with 10 mL 100% B. Hatched areas indicate elution position of the MAb. 2.0 Time (min) 5.0 0 A U 2 80 nm 3. 0 0 A U 2 80 nm 3. 0 a