Comparison of Camelid Antibody Ligand to Protein A for Monoclonal Antibody Purification - A stable alternative to Protein A chromatography. - BioPharm International


Comparison of Camelid Antibody Ligand to Protein A for Monoclonal Antibody Purification
A stable alternative to Protein A chromatography.

BioPharm International
Volume 22, Issue 9


As the newest alternative to Protein A chromatography, the camelid antibody ligand provided very good selectivity for IgG-based molecules. This resin showed similar binding affinity and somewhat comparable dynamic binding capacity to the commonly used commercial Protein A resins. For antibodies, it did have a lower capacity than the leading agarose-based Protein A resin; however, this shortcoming might be compensated by the vendor by further optimizing the ligand density and appropriate resin pricing. This resin does offer the distinct advantage of being base stable. Different from previously reported mixed mode and Protein A mimetic ligands, this ligand showed as good host cell protein clearance as the Protein A resins used for comparison. Although the new resin has been primarily marketed for IgG3 purification, the results presented in this study demonstrate that this can potentially be used very effectively for industrial MAb purification. Parameters that remain to be evaluated comprehensively before its widespread acceptance and use are column lifetime and viral clearance. Finally, the binding mechanism exploration studies indicated a difference in ligand–IgG interactions between the two ligands. For IgSelect, medium to high concentration of kosmotropic salt should be avoided in the background buffer to maximize antibody recovery during purification.


The authors would like to acknowledge the process biochemistry and analytical groups at Bristol-Myers Squibb for their analytical assay support.

Jia Liu, PhD, is a process engineer in process sciences downstream, John L. Hickey is associate director of process sciences downstream and project management, and Sanchayita Ghose, PhD, is the manager of the process sciences downstream group, all at Bristol Myers Squibb Company, Syracuse, NY, 315.431.7930,
Aaron Cheung is a chemical engineering student at the Massachusetts Institute of Technology.


1. Fahrner RL, Whitney DH, Vanderlaan M, Blank GS. Performance comparison of Protein A affinity-chromatography sorbents for purifying recombinant monoclonal antibodies. Biotechnol Appl Biochem. 1999;30:121–8.

2. Iyer H, Henderson F, Cunningham E, Webb J, Janson J, Berk C, Conley L. Considerations during development of a Protein A-based antibody purification process. BioPharm Int. 2002;15(1):14–20,53.

3. Brorson K, Brown J, Hamilton E, Stein KE. Identification of protein A media performance attributes that can be monitored as surrogates for retrovirus clearance during extended re-use. J Chromatogr A. 2003;989:155–63.

4. Ghose S, Allen M, Hubbard B, Brooks C, Cramer SM. Antibody variable region interactions with Protein A: Implications for the development of generic purification processes. Biotechnol Bioeng. 2005;92:665–73.

5. Hahn R, Bauerhansl P, Shiahara K, Wizniewski C, Tscheliessnig A, Jungbauer A. Comparison of protein A affinity sorbents II. Mass transfer properties. J Chromatogr A. 2005;1093:98–110.

6. Fahrner RL, Knudsen HL, Basey CD, Galan W, Feuerhelm D, Vanderlaan M, Blank GS. Industrial purification of pharmaceutical antibodies: development, operation, and validation of chromatography processes. Biotechnol Genet Eng Rev. 2001;18:301–27.

7. Shukla AA, Hubbard B, Tressel T, Guhan S, Low D. Downstream processing of monoclonal antibodies—application of platform approaches. J Chromatogr B. 2007;848:28–39.

8. Newcombe A, Cresswell C, Davies S, Watson K, Harris G, O'donovan K, Francis R. Optimised affinity purification of polyclonal antibodies from hyper immunized ovine serum using a synthetic Protein A adsorbent, MAbsorbent A2P. J Chromatogr B. 2005;814:209–15.

9. Terman DS, Bertram JH. Antitumor effects of immobilized protein A and staphylococcal products: linkage between toxicity and efficacy, and identification of potential tumoricidal reagents. Eur J Cancer Clin Oncol. 1985;21:1115–22.

10. Burton SC, Harding DRK. Hydrophobic charge induction chromatography: salt independent protein adsorption and facile elution with aqueous buffers. J Chromatogr A. 1998;814:71–81.

11. Guerrier GP, Schwartz W, Boschetti E. New method for the selective capture of antibodies under physiological conditions. Bioseparation. 2000;9:211–21.

12. Sengupta J, Sinha P, Mukhopadhyay C, Ray PK. Molecular modeling and experimental approaches toward designing a minimalist protein having Fc-binding activity of Staphylococcal Protein A. Biochem Biophys Res Commun. 1999;256:6–12.

13. Sinha P, Sengupta J, Ray PK. Functional mimicry of Protein A of Staphylococcus aureus by a proteolytically cleaved fragment. Biochem Biophys Res Commun. 1999;260:111–6.

14. Lowe CR, Burton SJ, Burton NP, Alderton WK, Pitts JM, Thomas JA. Designer dyes: 'biomimetic' ligands for the purification of pharmaceutical proteins by affinity chromatography. Trends Biotechnol. 1992;10:442–8.

15. Li R, Dowd V, Stewart DJ, Burton SJ, Lowe CR. Design, synthesis, and application of a Protein A mimetic. Nat Biotechnol. 1998;16:190–5.

16. Boschetti E, Judd D, Schwartz W, Tunon P. Hydrophobic charge induction chromatography. Genet Eng News. 2000;20:1–4.

17. Schwartz W, Judd D, Wysocki M, Guerrier L, Birck-Wilson E, Boschetti E. Comparison of hydrophobic charge induction chromatography with affinity chromatography on protein A for harvest and purification of antibodies. J Chromatogr A. 2001;908:251–63.

18. Teng SF, Sproule K, Hussain A, Lowe CR. A strategy for the generation of biomimetic ligands for affinity chromatography. Combinatorial synthesis and biological evaluation of an IgG binding ligand. J Mol Recognit. 1999;12:67–75.

19. Teng SF, Sproule K, Hussain A, Lowe CR. Affinity chromatography on immobilized "biomimetic" ligands: Synthesis, immobilization and chromatographic assessment of an immunoglobulin G-binding ligand. J Chromatogr B. 2000;740:1–15.

20. Huse K, Böhme H-J, Scholz GH. Purification of antibodies by affinity chromatography. J Biochem Biophys Methods. 2002;51:217–31.

21. Fassina G, Verdoliva A, Palombo G, Ruvo M, Cassani G. Immunoglobulin specificity of TG19318: a novel synthetic ligand for antibody affinity purification. J Mol Recognit. 1998;11:128–33.

22. Roque ACA, Taipa MA, Lowe CR. An artificial protein L for the purification of immunoglobulins and Fab fragments by affinity chromatography. J Chromatogr A. 2005;1064:157–67.

23. Ghose S, Hubbard B, Cramer SM. Protein Interactions in Hydrophobic Charge Induction Chromatography (HCIC). Biotechnol Prog. 2005;21:498–508.

24. Ghose S, Hubbard B, Cramer SM. Evaluation and comparison of alternatives to Protein A chromatography MImetic and hydrophobic charge induction chromatographic stationary phases. J Chromatogr A. 2006;1122:144–52.

25. Muyldermans S. Single domain camel antibodies: current status. Rev in Mol Biotechnol. 2001;74:277–302.

26. Van der Linden RHJ, Frenken LGJ, de Geus B, Harmsen MM, Ruuls RC, Stok W, et al. Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies. Biochim Biophys Acta. 1999;1431:37–46.

27. Verheesen P, ten Haaft MR, Lindner N, Verrips CT, de Haard JJW. Beneficial properties of single-domain antibody fragments for application in immunoaffinity purification and immuno-perfusion chromatography. Biochem Biophys Acta. 2003;1624:21–8.

28. Frenken LG, van der Linden RHJ, Hermans PW, Bos JW, Ruuls RC, de Geus B, Verrips CT. Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae. J Biotechnol. 2000;78:11–21.

29. Low D, O'Leary R, Pujar NS. Future of antibody purification. J Chromatogr B. 2007;848:48–63.

30. The Bio Affinity Company. Caustic Stable Human IgG Capture Ligand, Application Note. Available from:

31. The Bio Affinity Company. CapSelect caustic stable anti Human IgG affinity ligands, the Biotechnology Application Center. Application note;2004. Available from:

32. GE Healthcare. GE application note 28-9257-92 AA; 2007. Uppsala, Sweden.

33. Gagnon P. Purification tools for monoclonal antibodies. Tucson, AZ: Validated Biosystems Inc.; 1996.

34. Ghose S, Hubbard B, Cramer SM. Binding capacity differences for antibodies and Fc-fusion proteins on protein A chromatographic materials. Biotechnol Bioeng. 2007;96:768–79.

35. Jungbauer A, Hahn R. Engineering Protein A affinity chromatography. Curr Opin Drug Discov Devel. 2004;7:248–56.

36. Swinnen K, Krul A, Goidsenhoven IV, Tichelt NV, Roosen A, Houdt KV. Performance comparison of protein A affinity resins for the purification of monoclonal antibodies. J Chromatogr B. 2007;848:97–107.

37. Ghose S, McNerney T, Hubbard B. Process scale bioseparations for the biopharmaceutical industry. Boca Raton, FL: CRC Press; 2007.

38. Deisenhofer J. Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution. Biochemistry. 1981;20:2361–70.

blog comments powered by Disqus



GPhA Issues Statement on Generic Drug Costs
November 20, 2014
Amgen Opens Single-Use Manufacturing Plant in Singapore
November 20, 2014
Manufacturing Issues Crucial to Combating Ebola
November 20, 2014
FDA Requests Comments on Generic Drug Submission Criteria
November 20, 2014
USP Joins Chinese Pharmacopoeia Commission for Annual Science Meeting
November 20, 2014
Author Guidelines
Source: BioPharm International,
Click here