Evaluation and Understanding of a Chromatographic Media Ligand Density and its Impact on Process Performance in a Quality by Design World

Transcription

1 TM PERFORMACE MATERIALS PerformanceProfile Evaluation and Understanding of a Chromatographic Media Ligand Density and its Impact on Process Performance in a Quality by Design World by: Bhaktavachalam Thiyagarajan, Ph.D. Group Leader, Avantor Performance Materials Klaus Lohse Senior Application Engineer, Avantor Performance Materials andu Deorkar, Ph.D. Vice President, Pharmaceutical and Central R&D, Avantor Performance Materials The concept of Quality by Design (QbD) has been heavily promoted in the pharmaceutical and biotechnology industries in the past few years as an effective approach to ensure drug quality and patient safety. The primary focus of QbD is a thorough understanding of the manufacturing process, raw materials and product attributes. In this article, we present a study of the consistency of a raw material used in drug manufacturing and its impact on process performance. We evaluated the impact of ligand density on the separation performance and breakthrough capacity of BAKERBOD PolyPEI (hereafter PolyPEI), a polymer-based multimode weak anion exchange chromatography media. Samples of PolyPEI with percent nitrogen content meeting established specification range were compared to samples outside of the specification range. Both were synthesized and chromatographic separations were then performed. The results show that the retention time, separation efficiency and breakthrough capacity remained unchanged over the established specification range. The samples outside the specification range show decrease in separation efficiency while maintaining breakthrough capacity and retention time. The results indicate that PolyPEI offers lot-to-lot consistency and consistent separation performance. This type of evaluation can enable improved risk assessment and a better understanding of potential lot-to-lot variability, and its impact on processes. Introduction The concept of Quality by Design (QbD), while not new, has presented implementation opportunities and challenges to both the industry and regulators (1 4). Various experimental approaches and tools, such as design of experiments (DoE), cause and effect analysis, and multivariate analysis, have been used for systematic risk assessment and identification of critical quality attributes and critical process parameters (5 7). The intention of QbD is to ensure that manufacturing processes result in products that meet predefined parameters for product quality. Key elements in defining these parameters (quality profile) for a target product include: Understanding of raw material variability and its impact on process performance and product quality. Designing robust processes that are tolerant of slight variations to raw material attributes such as chromatographic media. Identifying critical quality attributes, process parameters and sources of variability. Controlling processes to produce consistent quality. At the center of downstream purification in the biopharmaceutical manufacturing processes, chromatographic media are critical raw materials in achieving a consistent process and ultimately in producing a quality product. For certain processes, traditional ion-exchangers often require tight control of critical process parameters such as conductivity and p (8). Lot-to-lot consistency of chromatographic media is critical to enabling and maintaining a robust operation so that there is a minimum change in separation performance, column efficiency and breakthrough capacity. In general, the variability in the performance of chromatographic media is due to variability in the ligand density and its impact on the separation performance. The objective of this study is to evaluate the impact of ligand density on separation efficiency and breakthrough capacity of BAKERBOD PolyPEI, PERFORMACE PROFILE

2 which is a polymer-based, multimode weak anion exchanger with a particle size of 35µ. The functionality of this product is obtained by covalently bonding PEI to the surface of highly cross-linked polymethacrylate beads. As illustrated in FIGURE 1, a unique ligand chemistry results in a variety of weak anionic exchange sites due to the presence of primary, secondary and tertiary amine groups on the polyethylenimine (PEI) ligands. Experimental Method Materials: PolyPEI (P 7585), tris base (tris (hydroxymethyl)- amino methane, P 419), sodium chloride (P 458) and sodium hydroxide (P 4722) were obtained from Avantor Performance Materials (Phillipsburg, J). Proteins BSA, IgG, lysozyme, ß-lactoglobulin-B and ß-lactoglobulin-A were obtained from Sigma and used without further purification. Separation and dynamic binding capacity were carried out using a packed column (7.75 x 1 cm) with Akta Explorer chromatography system from GE ealthcare. TABLE 1 shows specifications and average lot results for eight different lots of PolyPEI. The percent nitrogen measurement TABLE 1 Established PolyPEI Specifications and Typical Lot Result Averages. Test Parameter Specification Average 2 2 FIGURE 1 Ligand Structure of PolyPEI. Standard Deviation (SD) Particle Size 3 4µ /- 2 6 %C information only /- 3 6 % SD % (1.1 mm/ml) +/ IgG K 1 information only 2.3 +/ BSA, K 1 information only 6. +/ is conducted by carbon-hydrogen-nitrogen (C) analysis and indicates ligand density. In order to determine the impact of ligand density, samples of PolyPEI with nitrogen content outside of the established specification range (nitrogen percentage = 2.8, 3.9) were synthesized by using different concentrations of reagents and processes used in a typical production lot. Samples of commercial lots within specification, as well as special lots having nitrogen content slightly outside of the established specification, were used in this study. The percent nitrogen content specification range represents ligand density range of 1. to 1.4 millimoles per milliliter (mm/ml). All PolyPEI samples were packed in columns of 5 ml volume (.77 x 1 cm) for testing of separation performance. The selectivity/resolution was evaluated by injecting a sample consisting of five proteins with different isoelectric point (pi) and molecular weight (TABLE 2). TABLE 2 Mixture of Proteins with Different Isoelectric Point (pi) and Molecular Weight. Protein Sample Concentration (mg/ml) pi MW (kd) Lysozyme IgG, human 3. 7 BSA ß-Lactoglobulin-B ß-Lactoglobulin-A Chromatographic separations were performed by injecting 2 ml of protein mixture sample and running a linear gradient from to % buffer (B) in 1 column volumes (CV). (Buffer A: mm Tris, adjusted to p 8 using Cl; buffer B: buffer A with 1 M acl, p 8). Similarly, the Dynamic Binding Capacity (DBC) of media with various % was determined using Bovine Serum Albumin (BSA) as the model protein. Before each experiment, the columns were washed with 5 column volume (CV) of.5 ao at 3 ml/min. Binding buffer A: 2 mm sodium acetate, p 6.2, elution buffer B: 1 molar (M) sodium acetate, p 6.2, ml of 1 mg/ml BSA samples were injected at 2.4 ml/min (32 cm/hr). The bound protein was eluted using elution buffer B in 5 CV after washing with of 5 CV of buffer A. PERFORMACE PROFILE 2

3 Results and Discussion As illustrated in FIGURE 2 (a d), the retention times of PolyPEI that meet specification with nitrogen content between 4.5 and 6.5% are constant and within performance tolerances of the packed columns and chromatographic separation. Separation efficiency also remains unchanged. PolyPEI with a 2.8% content, which is below established specification range, show the retention times to differ by about 3% from other samples. Selectivity at 2.8% is maintained as shown by the insignificant change in retention time, however, decreased separation performance is observed below 3.9% as shown by decreased peak separation. The separation performance for model proteins is reproducible over the specified range of ligand density. 6.1% itrogen 5.3% itrogen ml FIGURE 2a Separation of lysozyme, IgG, BSA, ß-Lactoglobulin-B and ß-Lactoglobulin-A on PolyPEI with Various itrogen Contents ml FIGURE 2b 2.8% itrogen 3.9% itrogen ml FIGURE 2c ml FIGURE 2d PERFORMACE PROFILE 3

4 FIGURES 3 and 4 show that the breakthrough capacity of PolyPEI samples from established specification range (% = 6.1 and 4.4) and outside of that range (% = 3.9 and 2.8) do not change significantly with the change in ligand density. This consistent breakthrough capacity performance can be attributed to the high ligand density range of over.6 mmol/ml (% = 2.8). 8 6 Conclusion The above results show consistent selectivity and capacity of PolyPEI over the entire established specification range of 4.5 to 6.5%. This consistent performance is attributed to the availability of high ligand density. This consistent lot-to-lot performance of PolyPEI, therefore, should allow the design of a robust manufacturing process that does not require extremely tight control of critical process parameters and maintains performance when different lots of chromatographic media is used. This type of evaluation can help pharmaceutical and biotechnology manufacturers understand the impact of chromatographic media variability and incorporate the Quality by Design concept into their processes ml FIGURE 3 Breakthrough Curve of PolyPEI with a itrogen Content of 4.4% % DBC of BSA on PolyPEI (mg/ml) % itrogen FIGURE 4 DBC of PolyPEI with Various itrogen Content. PERFORMACE PROFILE 4

5 References 1. Pharmaceutical Quality for the 21st Century, A Risk-Based Approach Progress Report. Department of ealth and uman Services, U.S. Food and Drug Administration, May Presentation: umira Downstream Process: Challenges in Continuous Improvement and Technical Transfer, BioPharm International Interview with elen Yang, Abbott Research Center. 3. Angie Drakulich, Critical Challenges to Implementing QbD, A Q&A with FDA, Pharmaceutical Technology, October Susan Cook et al., Quality by Design in the CMO Environment, BioPharm International, December Amit Banerjee et. al., Designing in Quality: Approaches to Defining the Design Space for a Monoclonal Antibody Process, BioPharm International, May Wei Guo et al., Statistical Approach to IgG Binding on a Strong Cation Exchanger, BioProcess International, October Anurag Rathore et. al., Quality by Design: Industrial Case Studies on Defining and Implementing Design Space for Pharmaceutical Processes, BioPharm International, December Timothy M. Pabst et. al., Evaluation of Polyethyleneimne Based Chromatographic Support, ACS Poster (Spring ACS meeting 211, Anaheim, CA, USA). About the Authors Bhaktavachalam Thiyagarajan, Ph.D., is a Group Leader at Avantor Performance Materials. Klaus Lohse is Senior Application Engineer at Avantor Performance Materials. andu Deorkar, Ph.D., is Vice President, Pharmaceutical and Central R&D at Avantor Performance Materials. Phillipsburg, J 91: 28 & 141: 24 Paris, KY 91: 28 Mexico City, Mexico 91: 28 Deventer, the etherlands 91: 28 & 141: 24 & 13485: 23 Selangor, Malaysia 91: 28 About Avantor Performance Materials Avantor Performance Materials manufactures and markets high-performance chemistries and materials around the world under several respected brand names, including the J.T.Baker, Macron Fine Chemicals, Rankem, Diagnova, BeneSphera, and POC brands. Avantor products are used in a wide range of industries. Our biomedical and life science solutions are used in academic, industry and quality control laboratories for research, pharmaceutical production and medical lab testing, while our electronics solutions are used in the manufacturing of semiconductors and flat panel displays. Based in Center Valley, Pennsylvania (USA), Avantor is owned by an affiliate of ew Mountain Capital, LLC. For additional information please visit or follow Ordering Information and Assistance Customer Service and Technical Service toll free: AVATOR ( ) outside of u.s. tel: fax: info@avantormaterials.com AskAvantor Our Web site features ASK Avantor, which includes live chat capabilities with customer service representatives. Lit umber: Avantor Performance Materials, Inc. All rights reserved. Trademarks are owned by Avantor Performance Materials, Inc. or its affiliates unless otherwise noted. Corporate eadquarters Avantor Performance Materials, Inc Corporate Parkway Suite #2 Center Valley, PA 1834 USA Worldwide Locations China Malaysia orth America India Mexico Taiwan Korea The etherlands For contact information at these locations, visit Us/Worldwide-Directory.aspx