Application Note. USD 2995 (a) High Throughput Regeneration Study on MEP HyperCel Mixed-Mode Sorbent on ScreenExpert RoboColumns u

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1 Application Note USD 2995 (a) High Throughput Regeneration Study on MEP HyperCel Mixed-Mode Sorbent on ScreenExpert RoboColumns u

2 Summary A regeneration study of MEP HyperCel mixed-mode sorbent was conducted using 2 μl ScreenExpert RoboColumns operated with an automated Freedom EVO u workstation (Tecan). The study demonstrates the suitability of ScreenExpert RoboColumns for scale down evaluation of chromatographic operating parameters such as cleaning-in-place (CIP) conditions. This is of particular interest when different CIP regimens need to be evaluated to identify the best regeneration conditions on mixed-mode sorbents. The ScreenExpert RoboColumns miniaturized format offers significant advantages in saving feedstock and run time. 1. Introduction Mixed-mode sorbents are increasingly used in purification processes for biopharmaceutical productions. They offer unique selectivity for the separation of target proteins from contaminants. However, the use of mixed-mode sorbents require optimization of operating conditions, including CIP procedure, to achieve best performances, but CIP procedure optimization is time- and sample-consuming when performed on traditional column formats. In addition, the demand to reduce purification process development time and cost is increasing. To overcome these constraints, high throughput methods have been developed extensively for the screening of operating parameters using 96-well filter plates filled with chromatography sorbents. This format is well suited for the parallel evaluation of large numbers of conditions [1]. However, because filter plates are generally intended for single use, they are not suited for the screening of CIP conditions which requires repeated loading of feedstock and harsh washing steps [2]. ScreenExpert RoboColumns are minaturized chromatography columns of 2 and 6 μl operated on robotic liquid handling workstations [3]. They have a sorbent packed bed maintained between two filter frits. This design allows operation of these columns similarly to standard columns, and sorbent regeneration for repeated use as well. This application note describes a regeneration study to evaluate the lifetime performance of MEP HyperCel sorbent. Regeneration was performed using ScreenExpert RoboColumns on a Freedom EVO workstation. 5 purification cycles using.5 M NaOH for CIP were run. The dynamic binding capacity (DBC) was measured every 5 or 1 cycles. 2. Materials and Methods 2.1. Regeneration Runs 5 regeneration cycles were run on ScreenExpert RoboColumns (.5 x1. cm, 2 μl) packed with MEP HyperCel sorbent and another sorbent known for its low regeneration performance (called Negative control sorbent ). Every cycle, 75 mg of human polyclonal IgG (huigg) was loaded per ml of sorbent. The DBC at 1% breakthrough (BT) was measured on cycles No. 1, 5, 1, 15, 2, 25, 3, 4 and 5. Regeneration was performed in duplicate on two ScreenExpert RoboColumns for each sorbent using a Freedom EVO workstation equipped with a Te-Chrom u module and a 8-needle liquid handling head of 1 ml. The method was written using the Te-Chrom Wizard. 2

3 Table 1 Regeneration Sequence for One Cycle Step Buffer Residence Time Column Volumes Step Duration Equilibration PBS 1 min 1 1 min Load huigg 5 g/l in PBS 4 min 15 6 min Wash PBS 2 min 1 2 min Elution 5 mm Na acetate, ph 3. 2 min 1 2 min CIP.5 M NaOH 4 min min Reequilibration PBS 1 min 1 1 min 2.2. Determination of Fraction Volumes During fraction collection from ScreenExpert RoboColumns, the number of drops may slightly differ from well to well, inducing volume variations among the fractions [4]. Therefore, a method for measurement of fraction volumes was applied to determine the breakthrough volumes and the associated DBC accurately. The volume in each well was measured using a method adapted from McGown [5]. The liquid pathlength was determined by measuring the absorbance difference: Abs. 977 nm Abs. 9 nm. Then, the fraction volumes were calculated using calibration curves correlating volume to the pathlength for huigg solution in PBS. Fraction volume measurement and huigg quantitation were performed simultaneously. 3. Results 3.1. DBC Evaluation An example of the breakthrough curves obtained during the regeneration runs is presented in Figure 1. The curves follow a progressive breakthrough profile until a saturation plateau similarly to the typical behavior observed on standard columns. Figure 1 Determination of 1% BT Capacity on MEP HyperCel Sorbent and a Negative Control Sorbent on 2 μl ScreenExpert RoboColumns. Breakthrough Curves Obtained During the First Cycle MEP HyperCel Sorbent Negative Control Sorbent.7 C / Co DBC 1% BT: 17 mg/ml IgG Volume Loaded (ml) DBC 1% BT: 42 mg/ml 3

4 3.2. Regeneration Study The evolution of DBC at 1% BT through the 5 regeneration cycles is presented in Figure 2. MEP HyperCel sorbent provided a very stable DBC during the 5 cycles. In contrast, the negative control sorbent showed a significant capacity decrease after 15 cycles (8% decrease). Figure 2 Evolution of DBC at 1% BT on 5 Cycles on MEP HyperCel Sorbent and a Negative Control Sorbent 12 % of Initial DBC at 1% BT MEP HyperCel Sorbent Negative Control Sorbent Run Number.5 M NaOH, 3 minute CIP. Each data point is the mean value from two column measurements Data on ScreenExpert RoboColumns are similar to those usually obtained on prepacked columns Productivity of ScreenExpert RoboColumns The study performed on ScreenExpert RoboColumns provides significant benefits in terms of time and cost savings compared to standard laboratory columns. As presented in Figure 3, a ScreenExpert RoboColumns regeneration study requires 5-fold less protein for load because of the smaller column volume compared to 1 ml prepacked columns. In addition, up to 8 ScreenExpert RoboColumns can be operated in parallel with a Freedom EVO workstation, which drastically reduces run time and offers the possibility to run replicate columns and/or screen different conditions at the same time. Figure 3 Comparison of ScreenExpert RoboColumns and Laboratory Scale Columns (1 ml PRC Prepacked Columns) on Load Quantity and Run Duration for a Regeneration Study Load Quantity (g) / Run Duration (days) Load Quantity Run Duration ScreenExpert RoboColumn 1 ml PRC Prepacked Column 4

5 4. Conclusions The results from the regeneration study of MEP HyperCel sorbent performed with ScreenExpert RoboColumns were consistent with data usually obtained on 1 ml prepacked columns. Additionally, data showing a poor regeneration of another mixed-mode sorbent using prepacked columns were confirmed using ScreenExpert RoboColumns. This study demonstrates that ScreenExpert RoboColumns are well suited for high throughput regeneration study of mixed-mode sorbents using multiple CIP regimens. ScreenExpert RoboColumns are suitable tools for repeated CIP cycles. This format therefore greatly complements microplates for high throughput experiments and offers significant advantages over standard laboratory size columns with reduced consumption of feedstock and run time, leading to substantially improved productivity. References 1. Pall Application Note USD 294: High Throughput Chromatography Sorbent Screening and Purification Process Optimization of a Fab Antibody Fragment Using AcroPrep ScreenExpert Plates and PRC Prepacked Columns 2. Pall Instructions For Use USD 288: AcroPrep ScreenExpert plates 3. Pall Instructions For Use USD 2937: ScreenExpert RoboColumn 4. Treier, K, et al., High-Throughput Methods for Miniaturization and Automation of Monoclonal Antibody Purification Processes. Biotechnol. Prog. 212 May-Jun;28(3): McGown, E.L., Hafeman, D.G., Multichannel Pipettor Performance Verified by Measuring Pathlength of Reagent Dispensed Into a Microplate. Anal. Biochem. 1997;258: Corporate Headquarters Port Washington, NY, USA toll free (USA) phone biopharm@pall.com European Headquarters Fribourg, Switzerland +41 () phone LifeSciences.EU@pall.com Asia-Pacific Headquarters Singapore phone sgcustomerservice@pall.com Visit us on the Web at us at biopharm@pall.com International Offices Pall Corporation has offices and plants throughout the world in locations such as: Argentina, Australia, Austria, Belgium, Brazil, Canada, China, France, Germany, India, Indonesia, Ireland, Italy, Japan, Korea, Malaysia, Mexico, the Netherlands, New Zealand, Norway, Poland, Puerto Rico, Russia, Singapore, South Africa, Spain, Sweden, Switzerland, Taiwan, Thailand, the United Kingdom, the United States, and Venezuela. Distributors in all major industrial areas of the world. To locate the Pall office or distributor nearest you, visit The information provided in this literature was reviewed for accuracy at the time of publication. Product data may be subject to change without notice. For current information consult your local Pall distributor or contact Pall directly. 214, Pall Corporation Pall,, AcroPrep and HyperCel are trademarks of Pall Corporation. Filtration.Separation.Solution is a service mark of Pall Corporation. RoboColumn and RoboColumns are trademarks of Atoll GmbH. Freedom EVO and Te-Chrom are trademarks of Tecan Group. indicates a trademark registered in the USA 9/14, PDF, GN USD 2995(a)