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Implementation of Innovative Process Analytical Technologies to Characterize Critical Quality Attributes of Co‐Formulated Monoclonal Antibody Products

Godbole, Apurva, Chen, Lyufei, Desai, Jay, Raghava, Smita, Ruzanski, Richard, Patel, Bhumit, Appiah‐Amponsah, Emmanuel, Feng, Hanzhou·2025·Biotechnology and Bioengineering
DOI 10.1002/bit.28881
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Abstract

Characterizing co‐formulated monoclonal antibodies (mAbs) poses significant challenges in the pharmaceutical industry. Due to the high structural similarity of the mAbs, traditional analytical methods, compounded by the lengthy method development process, hinder product development and manufacturing efficiency. There is increasing critical need in the pharmaceutical industry to streamline analytical approaches, minimizing time and resources, ensuring a rapid clinical entry and cost‐effective manufacturing. This study investigates the application of process analytical technologies (PAT) to address such challenges. Our investigation introduces two complementary technologies, on‐line ultra‐performance liquid chromatography (online UPLC) and multimode fluorescence spectroscopy (MMFS), as potential PAT tools tailored for characterizing critical quality attributes (CQA) in co‐formulated mAb products. Specifically, the CQAs under evaluation include the total protein concentration of the mAbs within the co‐formulation and the ratio of mAb A to mAb B. Online UPLC enables direct and automated measurement of the CQAs through physical separation, while MMFS determines them in a non‐destructive and more swift manner based on chemometric modeling. We demonstrate these technologies' comparable performance to conventional methods, alongside substantial benefits such as reduced analytical turnaround time and decreased laboratory efforts. Ultimately, integrating them as innovative PAT tools expedites the delivery of therapeutic solutions to patients and enhances manufacturing efficiency, aligning with the imperative for swift translation of scientific discoveries into clinical benefits.

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  • The UPLC effectively distinguished and identified co-formulated antibodies despite their high structural similarity, which would have been challenging with other techniques such as spectroscopy.

    “The UPLC was able to effectively distinguish and identify the co‐formulated antibodies despite their high structural similarity [84] which would have been challenging with other techniques such as spectroscopy.”

    From Current PAT Landscape in the Downstream Processing of Biopharmaceuticals (2025) · cites this paper as [84]

  • On-line UPLC took 30 min of measurement time per sample, highlighting the need for speeding up separation time to use chromatography as a realistic PAT tool.

    “Though this significantly reduced the analytical turnaround time, it still took 30 min of measurement time per sample [84], highlighting the need for speeding up the separation time to be able to use chromatography as a realistic PAT tool.”

    From Current PAT Landscape in the Downstream Processing of Biopharmaceuticals (2025) · cites this paper as [84]

  • Godbole et al. in 2024 used on-line UPLC based on ion exchange to determine the ratio of co-formulated mAbs.

    “An on‐line ultra‐performance liquid chromatography (UPLC), based on ion exchange, had been employed by Godbole et al., in 2024 to determine the ratio of co‐formulated mAbs.”

    From Current PAT Landscape in the Downstream Processing of Biopharmaceuticals (2025) · cites this paper as [84]

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Research works printed but not linked: 30

  1. Abu-Absi, N. R., B. M. Kenty, M. E. Cuellar, et al. 2011. "Real Time Monitoring of Multiple Parameters in Mammalian Cell Culture Bioreactors Using an In-Line Raman Spectroscopy Probe." Biotechnology and Bioengineering 108, no. 5: 1215-1221. https://doi.org/10.1002/bit.23023. · DOI · Paper page
  2. Alvarez, M., G. Tremintin, J. Wang, et al. 2011. "On-Line Characterization of Monoclonal Antibody Variants by Liquid Chromatography-Mass Spec- trometry Operating in a Two-Dimensional Format." Analytical Biochemistry 419, no. 1: 17-25. https://doi.org/10.1016/j.ab.2011.07.033. · DOI
  3. André, S., L. S. Cristau, S. Gaillard, O. Devos, É. Calvosa, and L. Duponchel. 2015. "In-Line and Real-Time Prediction of Recombinant Antibody Titer by in Situ Raman Spectroscopy." Analytica Chimica Acta 892: 148-152. https://doi.org/10.1016/j.aca.2015.08.050. · DOI
  4. Bera, G., G. Gold-Bouchot, U. Passow, et al. 2019. "Inter-Laboratory Calibration of Estimated Oil Equivalent (EOE) Concentrations of a Water Accommodated Fraction (WAF) of Oil and a Chemically En- hanced Waf (CEWAF)." Heliyon 5, no. 1: e01174. https://doi.org/10. 1016/j.heliyon.2019.e01174. · DOI
  5. Bergin, A., J. Carvell, and M. Butler. 2022. "Applications of Bio- Capacitance to Cell Culture Manufacturing." Biotechnology Advances 61: 108048. https://doi.org/10.1016/j.biotechadv.2022.108048. · DOI
  6. Camperi, J. 2022. "Online HPLC-HRMS Platform: The Next-Generation Process Analytical Technology Tool for Real-Time Monitoring of An- tibody Quality Attributes in Biopharmaceutical Processes." LCGC North America 1: 20-22,29. https://doi.org/10.56530/lcgc.na.op5766f2. · DOI
  7. Dos Santos Rodrigues, F. H., G. G. Delgado, T. Santana da Costa, and L. Tasic. 2023. "Applications of Fluorescence Spectroscopy in Protein Conformational Changes and Intermolecular Contacts." BBA Advances 3: 100091. https://doi.org/10.1016/j.bbadva.2023.100091. · DOI
  8. Dunn, Z. D., P. Bohman, A. Quinteros, et al. 2023. "Automated Online- Sampling Multidimensional Liquid Chromatography with Feedback- Control Capability as a Framework for Real-Time Monitoring of mAb Critical Quality Attributes in Multiple Bioreactors." Analytical Chemistry 95: 18130-18138. https://doi.org/10.1021/acs.analchem. 3c03528. · DOI
  9. Feng, H., Z. D. Dunn, and R. Kargupta, et al. 2024. "Pioneering Just-in- Time (JIT) Strategy for Accelerating Raman Method Development and Implementation for Biologic Continuous Manufacturing." Analytical Chemistry. Published ahead of print, February 6, 2024. https://doi.org/ 10.1021/acs.analchem.3c05628. · DOI
  10. Harshman, R. A., and M. E. Lundy. 1994. "PARAFAC: Parallel Factor Analysis." Computational Statistics & Data Analysis 18, no. 1: 39-72. https://doi.org/10.1016/0167-9473(94)90132-5. · DOI
  11. Jiang, M., K. A. Severson, J. C. Love, et al. 2017. "Opportunities and Challenges of Real-Time Release Testing in Biopharmaceutical Manu- facturing." Biotechnology and Bioengineering 114, no. 11: 2445-2456. https://doi.org/10.1002/bit.26383. · DOI · Paper page
  12. Kim, J., Y. J. Kim, M. Cao, et al. 2020. "Analytical Characterization of Coformulated Antibodies as Combination Therapy." MAbs 12, no. 1: 1738691. https://doi.org/10.1080/19420862.2020.1738691. · DOI
  13. Kim, Y. K., N. P. Illsley, and A. S. Verkman. 1988. "Rapid Fluorescence Assay of Glucose and Neutral Solute Transport Using an Entrapped Volume Indicator." Analytical Biochemistry 172, no. 2: 403-409. https:// doi.org/10.1016/0003-2697(88)90462-9. · DOI
  14. Kinch, M. S., Z. Kraft, and T. Schwartz. 2023. "Monoclonal Antibodies: Trends in Therapeutic Success and Commercial Focus." Drug Discovery Today 28, no. 1: 103415. https://doi.org/10.1016/j.drudis.2022.103415. · DOI
  15. Köhler, G., and C. Milstein. 1975. "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity." Nature 256, no. 5517: 495-497. https://doi.org/10.1038/256495a0. · DOI
  16. Krieg, D., G. Winter, and H. L. Svilenov. 2022. "It Is Never Too Late for a Cocktail -Development and Analytical Characterization of Fixed- Dose Antibody Combinations." Journal of Pharmaceutical Sciences 111, no. 8: 2149-2157. https://doi.org/10.1016/j.xphs.2022.05.014. · DOI
  17. Lakowicz, J. R. 2006. Principles of Fluorescence Spectroscopy. New York: Springer.
  18. Li, Y., D. Hewitt, Y. K. Lentz, J. A. Ji, T. Y. Zhang, and K. Zhang. 2014. "Characterization and Stability Study of Polysorbate 20 in Therapeutic Monoclonal Antibody Formulation by Multidimensional Ultrahigh- Performance Liquid Chromatography-Charged Aerosol Detection-Mass Spectrometry." Analytical Chemistry 86, no. 10: 5150-5157. https://doi. org/10.1021/ac5009628. · DOI
  19. Lyu, X., Q. Zhao, J. Hui, et al. 2022. "The Global Landscape of Approved Antibody Therapies." Antibody Therapeutics 5, no. 4: 233-257. https:// doi.org/10.1093/abt/tbac021. · DOI
  20. Maria Tchamova, J. F. 2021. "Evaluation of the Slope Spectroscopy Method for Monoclonal Antibodies." American Pharmaceutical Review 1: 1-6.
  21. Murphy, K. R., C. A. Stedmon, D. Graeber, and R. Bro. 2013. "Fluo- rescence Spectroscopy and Multi-Way Techniques. Parafac." Analytical Methods 5, no. 23: 6557. https://doi.org/10.1039/c3ay41160e. · DOI
  22. Noble, J. E., and M. J. Bailey. 2009. "Quantitation of Protein." Methods in Enzymology 463: 73-95. https://doi.org/10.1016/s0076-6879(09) 63008-1. · DOI · Paper page
  23. Patel, B. A., N. D. S. Pinto, A. Gospodarek, et al. 2017. "On-Line Ion Exchange Liquid Chromatography as a Process Analytical Technology for Monoclonal Antibody Characterization in Continuous Bioproces- sing." Analytical Chemistry 89, no. 21: 11357-11365. https://doi.org/10. 1021/acs.analchem.7b02228. · DOI
  24. Quatela, A., A. M. Gilmore, K. E. S. Gall, et al. 2018. "A-TEEM(TM), a New Molecular Fingerprinting Technique: Simultaneous Absorbance- Transmission and Fluorescence Excitation-Emission Matrix Method." Methods and Applications in Fluorescence 6, no. 2: 027002. https://doi. org/10.1088/2050-6120/aaa818. · DOI
  25. Rhoades, E., E. Gussakovsky, and G. Haran. 2003. "Watching Proteins Fold One Molecule at a Time." Proceedings of the National Academy of Sciences 100, no. 6: 3197-3202. https://doi.org/10.1073/pnas. 2628068100. · DOI
  26. Schwab, K., T. Amann, J. Schmid, R. Handrick, and F. Hesse. 2016. "Exploring the Capabilities of Fluorometric Online Monitoring on Chinese Hamster Ovary Cell Cultivations Producing a Monoclonal Antibody." Biotechnology Progress 32, no. 6: 1592-1600. https://doi.org/ 10.1002/btpr.2326. · DOI
  27. Strohl, W. R. 2024. "Structure and Function of Therapeutic Antibodies Approved by the US FDA in 2023." Antibody Therapeutics 7, no. 2: 132- 156. https://doi.org/10.1093/abt/tbae007. · DOI
  28. Verkhusha, V. V., and K. A. Lukyanov. 2004. "The Molecular Properties and Applications of Anthozoa Fluorescent Proteins and Chromo- proteins." Nature Biotechnology 22, no. 3: 289-296. https://doi.org/10. 1038/nbt943. · DOI
  29. Weitner, T., T. Friganović, and D. Šakić. 2022. "Inner Filter Effect Correction for Fluorescence Measurements in Microplates Using Vari- able Vertical Axis Focus." Analytical Chemistry 94, no. 19: 7107-7114. https://doi.org/10.1021/acs.analchem.2c01031. · DOI
  30. Wu, S., S. A. Ketcham, C. C. Corredor, D. Both, J. K. Drennen, and C. A. Anderson. 2022. "Rapid At-Line Early Cell Death Quantification Using Capacitance Spectroscopy." Biotechnology and Bioengineering 119, no. 3: 857-867. https://doi.org/10.1002/bit.28011. · DOI

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Authors
Godbole, A., Chen, L., Desai, J., Raghava, S., Ruzanski, R., Patel, B., Appiah‐Amponsah, E., & Feng, H.
Affiliations
  • University of Illinois at Urbana‐Champaign (UIUC) Champaign Illinois USA
  • Horiba Instruments Incorporated Piscataway New Jersey USA
  • Data Rich Measurements Analytical Enabling Capabilities. Merck & Co., Inc. Rahway New Jersey USA
  • Sterile and Specialty Products Preclinical Development. Merck & Co., Inc. Rahway New Jersey USA
  • Biologics Analytical Research and Development Merck & Co., Inc. Rahway New Jersey USA
ORCID
Feng, Hanzhou
Venue
Biotechnology and Bioengineering
Publisher
Wiley
Published
2024-11-07
Volume
122
Issue
2
Pages
322-332
ISSN
  • 0006-3592
  • 1097-0290
DOI
10.1002/bit.28881
Cited by
4
References
1
Language
en
Licence
onlinelibrary.wiley.com/termsAndConditions#vor (vor)

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