Acta Scientific Medical Sciences (ISSN: 2582-0931)

Research Article Volume 5 Issue 10

Leveraging the Power of High-throughput Technologies for Chromatographical Purification

Dilara Baş1, Emre Burak Erkal1, Ali Özhan Aytekin2, Deniz Demirhan1,3*

1Biotechnology Group, Turgut Pharmaceuticals, İstanbul, Turkey
2Genetics and Bioengineering Department, Faculty of Engineering, Yeditepe University, Atasehir, İstanbul, Turkey
3Department of Natural Sciences, Faculty of Engineering, Acibadem Mehmet Ali Aydinlar University, Istanbul, Turkey

*Corresponding Author: Deniz Demirhan, Turgut Pharmaceuticals Biotechnology Group/Department of Natural Sciences, Faculty of Engineering, Acibadem Mehmet Ali Aydınlar University, İstanbul, Turkey.

Received:July 28, 2021 Published: September 24, 2021

Citation: Deniz Demirhan., et al. “Indirect (Mirror) Laryngoscopy: Valuable Laryngological Skill Going into Extinction?". Acta Scientific Medical Sciences 5.10 (2021): 144-155.


  Therapeutic monoclonal antibodies (mAbs) are target-specific proteins used for patients especially suffering from cancer, autoimmune, and inflammatory diseases. Due to the success rate of the mAbs, they are the fastest-growing sector in the biopharmaceutical industry. To be used for the therapeutic purposes, the monoclonal antibodies have to be high purity. A variety of different downstream technologies have recently been evolved to provide high quality monoclonal antibody and biosimilar development with low cost in a shorter time. Recent advancements of high-throughput process (HTP) technology are employed for process development for saving time, investment, and cost. HTP allows running large number of experiments in a minimal scale. Incorporation of HTP technology with Design of Experiment (DoE) is systematic integration for effective and rapid screening of optimum experimental conditions.

  Development of purification process for mAbs requires the consideration of many different parameters including removal of impurities and ready availability of raw materials for large-scale production. In this study, a downstream process was developed for one of the best-selling biosimilar mAb (TUR02) coupling HTP technology with DoE tools. TUR02 is an IgG1 which has a high tendency to aggregate. In order to develop protein A chromatography, firstly appropriate elution buffer had been selected using micro-volume columns. Large range of parameters were screened as buffer type (acetic acid, citric acid, sodium acetate), the concentration of buffer (10 mM-150 mM), and three different Protein A resins. Citric acid (10mM) at pH 3.5 was chosen as suitable elution buffer. A resin with base matrix of methacrylic polymer was chosen from the HTP study and a lab scale experiment with 1 ml column was done to prove the success of the scalability of the HTP technology. After protein A chromatography, HTP technology was applied to cation exchange chromatography to screen different conditions to be able to further decrease the impurity level. Flow through with overloading mode was followed with the cation exchange screening. Concentration of sodium phosphate-based buffer (10 mM – 50 mM), working pH and comparison of strong and weak cation exchange resin were screened by coupling HTP technology and DoE tools. Strong cation exchange resin, 38 mM sodium phosphate and pH 4.5 were selected due to its impurity reduction strength. Scalability of the process was successfully proven using 1 ml pre-packed column. With these conditions, the aggregate and HCP levels were 0.52 and 612 and 0.32 and 234 for 0.1 ml and 1 ml columns, respectively.

Keywords: Monoclonal Antibodies; Chromatography; High-Throughput Technology; Design of Experiment; Protein A Ligand; Cation Exchange


  1. Gronemeyer Petra., et al. “Trends in Upstream and Downstream Process Development for Antibody Manufacturing”. Bioengineering4 (2014): 188-212.
  2. Xu Zhihao., et al. “Process Development For Robust Removal Of Aggregates Using Cation Exchange Chromatography In Monoclonal Antibody Purification With Implementation Of Quality By Design”. Preparative Biochemistry and Biotechnology2 (2012): 183-202.
  3. Treier Katrin., et al. “High-Throughput Methods for Miniaturization and Automation of Monoclonal Antibody Purification Processes”. Biotechnology Progress3 (2012): 723-732.
  4. Shukla Abhinav A and Peter Hinckley. “Host Cell Protein Clearance during Protein a Chromatography: Development of an Improved Column Wash Step”. Biotechnology Progress5 (2008): 1115-1121.
  5. Liu, Hui F., et al. “Recovery and Purification Process Development for Monoclonal Antibody Production”. MAbs5 (2010): 480-499.
  6. Kelley Brian. “Downstream Processing Of Monoclonal Antibodies: Current Practices And Future Opportunities”. Process Scale Purification of Antibodies (2017): 1-21.
  7. Mazzer Alice R., et al. “Protein A Chromatography Increases Monoclonal Antibody Aggregation Rate during Subsequent Low PH Virus Inactivation Hold”. Journal of Chromatography A 1415 (2015): 83-90.
  8. Mahler Hanns-Christian., et al. “Protein Aggregation: Pathways, Induction Factors and Analysis”. Journal of Pharmaceutical Sciences9 (2009): 2909-2934.
  9. Liu Hui F., et al. “Recovery and Purification Process Development for Monoclonal Antibody Production”. MAbs5 (2010): 480-499.
  10. Thermo Scientific. Optimize Elution Conditions for Immunoaffinity Purification, TECH TIP # 27 / TR0027.1, (2009).
  11. GE Healthcare. Mahler Hanns-Christian., et al. “Protein Aggregation: Pathways, Induction Factors and Analysis”. Journal of Pharmaceutical Sciences9, 2009): 2909-2934.
  12. Plain & Crosslinked Agarose Bead Informational, FM-000008 / TD-I Revision 2.0 (2019).
  13. Kumar A., et al. “Polymer Displacement/Shielding in Protein Chromatography”. Journal of Chromatography B: Biomedical Sciences and Applications2 (2000): 103-113.
  14. Nogal Bartek., et al. “Select Host Cell Proteins Coelute with Monoclonal Antibodies in Protein a Chromatography”. Biotechnology Progress2 (2012): 454-458.
  15. Hober Sophia., et al. “Protein A Chromatography for Antibody Purification”. Journal of Chromatography B1 (2007): 40-47.
  16. Nogal Bartek., et al. “Select Host Cell Proteins Coelute with Monoclonal Antibodies in Protein a Chromatography”. Biotechnology Progress2 (2012): 454-458.
  17. Connell-Crowley Lisa., et al. “Cation Exchange Chromatography Provides Effective Retrovirus Clearance for Antibody Purification Processes”. Biotechnology and Bioengineering1 (2011): 157-165.
  18. Vázquez-Rey María and Dietmar A Lang. “Aggregates in Monoclonal Antibody Manufacturing Processes”. Biotechnology and Bioengineering7 (2011): 1494-1508.
  19. McCaw Tyler R., et al. “Evaluation of a Novel Methacrylate‐Based Protein a Resin for the Purification of Immunoglobulins and Fc‐Fusion Proteins”. Biotechnology Progress5 (2014): 1125-1136.
  20. “Ion Exchange Chromatography”. Bio.
  21. Flatman Stephen., et al. “Process Analytics for Purification of Monoclonal Antibodies”. Journal of Chromatography B1 (2007): 79-87.
  22. Buchner Johannes., et al. “Alternatively Folded States of an Immunoglobulin”. Biochemistry 28 (1991): 6922-6929.
  23. Sharma Basant. “Immunogenicity of Therapeutic Proteins. Part 3: Impact of Manufacturing Changes”. Biotechnology Advances3 (2007): 325-331.
  24. Tan Zhijun., et al. “On-Column Disulfide Bond Formation of Monoclonal Antibodies during Protein A Chromatography Eliminates Low Molecular Weight Species and Rescues Reduced Antibodies”. MAbs 1 (2020): 1829333.
  25. Mahler Hanns-Christian., et al. “Induction and Analysis of Aggregates in a Liquid IgG1-Antibody Formulation”. European Journal of Pharmaceutics and Biopharmaceutics3 (2005): 407-417.

Copyright: © 2021 Deniz Demirhan., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

News and Events

  • Certification for Review
    Acta Scientific certifies the Editors/reviewers for their review done towards the assigned articles of the respective journals.
  • Submission Timeline for Upcoming Issue
    The last date for submission of articles for regular Issues is April 30th, 2024.
  • Publication Certificate
    Authors will be issued a "Publication Certificate" as a mark of appreciation for publishing their work.
  • Best Article of the Issue
    The Editors will elect one Best Article after each issue release. The authors of this article will be provided with a certificate of "Best Article of the Issue".
  • Welcoming Article Submission
    Acta Scientific delightfully welcomes active researchers for submission of articles towards the upcoming issue of respective journals.

Contact US