October 15, 2018 (Vol. 38, No. 18)
Jacquelynn Smith Scientist Pfizer
Matthew A. Lauber Ph.D. Consulting Scientist Waters
Waters Enables High-Throughput, Efficient, and Reliable Characterization of mAbs and ADCs
Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are the fastest growing class of therapeutic agents. There are nearly 50 mAb-related products approved by the U.S. FDA and the European Medicines Agency (EMA) for the treatment of cancer and cardiovascular, neurological, autoimmune, and inflammatory diseases.1
Efficient and reliable detection and characterization of microheterogeneities in therapeutic mAbs and ADCs is of critical importance, as sources of variability inherent to the development and manufacturing process have the potential to affect the quality of the final product.1 For Pfizer, improving efficiency and reliability in the process of characterizing protein therapeutics is of critical importance. To help the company achieve its required benchmarks, scientists from Waters took on the challenge of engineering a novel reversed-phase liquid chromatography (RPLC) column for high-throughput separations with enhanced resolution, selectivity, and recovery of target mAbs and ADCs.
Quality Is in the Details
Unlike small molecule drugs, therapeutic mAbs are large, complex biomolecules, typically produced in mammalian cells through recombinant DNA technology. Several factors, including differences between cell lines, or between different clones derived from the same cell line, and variability in post-translational modifications during expression can result in batch-to-batch variability in the final product (Figure 1).2 Some degree of microheterogeneity can be tolerated by patients and is inherent to the process of mAb development, similar to the natural variability occurring in all biomolecules. However, to ensure consistency in the quality of the final product, a set of predefined critical quality attributes that have the potential to influence the quality, safety, and efficacy of a given biologic have to be monitored closely and kept within a certain limit, range, or distribution.
Quality risk assessment throughout the product development lifecycle is recommended by most international regulatory agencies in accordance with Quality by Design (QbD) principles.3–7 Effective implementation of QbD requires a thorough understanding of how variations in the chemical, biophysical, and microbiological properties of the biologic can affect product performance and require appropriate analytical methods that allow efficient and reliable monitoring of such variations. For companies like Pfizer, quality control is critical for product performance and integrity, and most importantly, to maintain the highest standards of patient safety. For instance, variations in post-translational modifications leading to heterogeneities in glycosylation or charge-variance patterns have the potential to affect the safety, efficacy, pharmacokinetic, and immunogenicity profile of a mAb or ADC (Figure 1).8
RPLC is a separation technique commonly used to characterize protein therapeutics during their development.9 It is a frequently-relied-upon methodology, due to its high resolving power and amenability to mass spectrometric (MS) detection.9 However, current RPLC columns are associated with performance limitations, including the dependence of the stationary phase on ion pairing and elevated temperatures, which can degrade mAbs, excessive carry-over of sample residue between successive separations, and the generation of unreliable results due to intercolumn irreproducibility.10
Waters responded to the need for more efficient and reliable methods for the separation and characterization of protein therapeutics by assembling a multidisciplinary team of research scientists tasked with engineering a column that would raise the standards for separation performance.
A Novel RPLC Column for Reliable Characterization of Protein Therapeutics
BioResolve™ RP mAb Polyphenyl Columns were designed specifically to address a multitude of technological challenges that are encountered during the characterization of intact mAbs or their subunits by RPLC, including the need for more efficient, higher resolution separations.10 Scientists from Waters worked to identify the parameters that would lead to enhanced resolution and selectivity, with limited degradation and carry-over, thereby allowing for higher quality, reproducible data. By combining the kinetic benefits of solid-core particle technology with a novel, high-coverage polyphenyl surface chemistry, BioResolve RP mAb Polyphenyl Columns allow users to perform higher throughput, reproducible separations, with enhanced resolution and selectivity for both RPLC and MS-compatible RPLC separations, at lower temperatures and with less acidic ion pairing to limit the degradation of target mAbs (Figures 2 & 3).10
Engineering a column that addresses the most pressing challenges in reversed-phase separation technology required a multidisciplinary team of research scientists working their way through nearly 200 prototypes over a period of three years. A close partnership with scientists at Pfizer’s Analytical R&D Mass Spectrometry and Biophysical Characterization Group was integral to the success of the project and ensured the end result would bring maximum value to the biopharmaceutical scientists working with mAbs and ADCs.
Together, Waters and Pfizer analyzed a range of mAbs and ADCs using different mobile-phase conditions.10–12 The results of these studies consistently demonstrated the superior kinetic properties of the base particle compared to conventional RPLC stationary phases, and showed that the novel, polyphenyl-bonded phase provides enhanced resolution and selectivity at a variety of temperature and ion-pairing conditions (Figure 3).11,12 Using BioResolve RP mAb Polyphenyl Columns, Pfizer scientists were able to successfully separate, recover, and subsequently characterize multiple mAbs and ADCs, including an ADC that had previously presented substantial challenges for an analysis with conventional RPLC stationary phases, due to a highly hydrophobic subunit.12
Pfizer’s Analytical R&D Mass Spectrometry and Biophysical Characterization group has incorporated BioResolve RP mAb Polyphenyl Columns in its standard operating procedures for the characterization of many mAbs and bioconjugates within the company’s pipeline, and have recognized substantial, tangible successes. Overall, use of the BioResolve RP mAb Polyphenyl Columns continues to provide high recovery, high-resolution separations for right-first-time analyses without extensive method development. Pfizer scientists and teams can be more confident that the respective protein and conjugate forms are closely monitored in terms of molecular integrity and/or extent of conjugation for assessment of product quality, safety, and efficacy.
Jacquelynn Smith is a scientist at the analytical R&D and biophysical characterization group at Pfizer. Matthew A. Lauber, Ph.D., is a consulting scientist at the Chemistry Technology Center at Waters.
1. Wang, X., An, Z., Luo, W., Xia, N. & Zhao, Q. Molecular and functional analysis of monoclonal antibodies in support of biologics development. Protein Cell 9, 74-85 (2018).
2. Liu H, Gaza-Bulseco, G. & Faldu, D. Heterogeneity of monoclonal antibodies. J Pharm Sci 97, 2426–2447 (2007).
3. Q8, I. Pharmaceutical Development, Step 4. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. (Geneva, Switzerland, 2009).
4. Q9, I. Quality Risk Management, Step 4. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. (Geneva, Switzerland, 2005).
5. Q10, I. Pharmaceutical Quality System, Step 4. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. (Geneva, Switzerland, 2008).
6. Q11, I. Development and Manufacture of Drug Substance — Chemical Entities and Biotechnological/Biological Entities, Step 4. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. (Geneva, Switzerland, 2012).
7. Yu, L.X., et al. Understanding pharmaceutical quality by design. AAPS J 16, 771-783 (2014).
8. Zhang, L., et al. Analysis of Monoclonal Antibody Sequence and Post-translational Modifications by Time-controlled Proteolysis and Tandem Mass Spectrometry. Mol Cell Proteomics 15, 1479-1488 (2016)
9. Wakankar, A., Chen, Y., Gokarn, Y. & Jacobson, F.S. Analytical methods for physicochemical characterization of antibody drug conjugates. MAbs 3, 161-172 (2011).
10. Bobaly, B., Lauber, M., Beck, A., Guillarme, D. & Fekete, S. Utility of a high coverage phenyl-bonding and wide-pore superficially porous particle for the analysis of monoclonal antibodies and related products. J Chromatogr A 1549, 63-76 (2018).
11. Nguyen J, Rzewuski S & D, W. A Novel Phenyl-Based RPLC Stationary Phase for High Throughput, High Resolution Characterization of Protein Therapeutics. in 22nd Symposium on the Interface of Regulatory and Analytical Sciences for Biotechnology Health Products (2018).
12. Smith J, Friese O.V & Rouse J.C. High Resolution Chromatography – Mass Spectrometry with a Novel Phenyl RPLC Column for Heightened Characterization of Hydrophobic Monoclonal Antibodies and Antibody Drug Conjugates. In 22nd Symposium on the Interface of Regulatory and Analytical Sciences for Biotechnology Health Products (Washington, DC, 2018).