Octanoic acid (OA) precipitation has long been used in protein purification. Recently, we reported a new cell culture clarification method for immunoglobulin G (IgG) purification, employing an advance elimination of chromatin heteroaggregates with a hybrid OA-solid phase system. This treatment reduced DNA more than 3 logs, histone below the detection limit (LOD), and non-histone host cell proteins (nh-HCP) by 90 % while conserving more than 90 % of the IgG monomer. In this study, we further investigated the conservation of IgG monomer and antibody light chain (LC) to the addition of OA/OA-solid phase complex, with or without histone and DNA in different combinations. The results showed that highly basic histone protein was the prime target in OA/OA-solid phase precipitation system for IgG purification, and the selective conservation of IgG monomer in this system was histone dependent. Our findings partially support the idea that OA works by sticking to electropositive hydrophobic domains on proteins, reducing their solubility, and causing them to agglomerate into large particles that precipitate from solution. Our findings also provide a new perspective for IgG purification and emphasize the necessity to re-examine the roles of various host contaminants in IgG purification.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Bhoskar P, Belongia B, Smith R, Yoon S, Carter T, Jin X (2013) Free light chain content in culture media reflects recombinant monoclonal antibody productivity and quality. Biotechnol Prog 29:1131–1139. doi:10.1002/btpr.1767
Birch JR, Racher AJ (2006) Antibody production. Adv Drug Deliv Rev 58:671–685. doi:10.1016/j.addr.2005.12.006
Brodsky Y, Zhang C, Yigzaw Y, Vedantham G (2012) Caprylic acid precipitation method for impurity reduction: an alternative to conventional chromatography for monoclonal antibody purification. Biotechnol Bioeng 109:2589–2598. doi:10.1002/bit.24539
Budavari S, O’Neil MJ, Smith A, Heckelman PE, Kinneary JF (1996) The Merck index: an encyclopedia of chemicals, drugs, and biologicals (12th ed.) Merck & Co. ISBN 0911910123
Chusainow J, Yang YS, Yeo JH, Toh PC, Asvadi P, Wong NS, Yap MG (2009) A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng 102:1182–1196. doi:10.1002/bit.22158
Gagnon P, Nian R, Lee J, Tan L, Abdul Latiff SM, Lim CL, Chuah C, Yang YS, Gan HT (2014a) Nonspecific interactions of chromatin with immunoglobulin G and protein A, and their impact on purification performance. J Chromatogr A 1340:68–78. doi:10.1016/j.chroma.2014.03.010
Gagnon P, Nian R, Tan L, Cheong J, Yeo V, Yang YS, Gan HT (2014b) Chromatin-mediated depression of fractionation performance on electronegative multimodal chromatography media, its prevention, and ramifications for purification of immunoglobulin G. J Chromatogr A 1374:145–155. doi:10.1016/j.chroma.2014.11.052
Gagnon P, Nian R, Yang YS, Yang Q, Lim CL (2015) Non-immunospecific association of immunoglobulin G with chromatin during elution from protein a inflates host contamination, aggregate content, and antibody loss. J Chromatogr A 1408:151–160. doi:10.1016/j.chroma.2015.07.017
Gao QS, Sun M, Tyutyulkova S, Webster D, Rees A, Tramontano A, Massey RJ, Paul S (1994) Molecular cloning of a proteolytic antibody light chain. J Biol Chem 269:32389–32393
Georgel PT, Hansen JC (2001) Linker histone function in chromatin: dual mechanisms of action. Biochem Cell Biol 79:313–316. doi:10.1139/o01-080
Haasa J, Fettinga F, Plogb C, Kerfinb W, Gerhardb W, Rothb G (2006) Recognition and classification of histones using support vector machine. J Comput Biol 13:102–112. doi:10.1089/cmb.2006.13.102
Hamilton RG (1990) 5—Production and epitope location of monoclonal antibodies to the human IgG subclasses. The Human IgG Subclasses 33:79–91. doi:10.1016/B978-0-08-037504-5.50010-7
Herzer S, Bhangale A, Barker G, Chowdhary I, Conover M, O’Mara BW, Tsang L, Wang SY, Krystek SR Jr, Yao Y, Rieble S (2015) Development and scale-up of the recovery and purification of a domain antibody fc fusion protein-comparison of a two and three-step approach. Biotechnol Bioeng 112:1417–1428. doi:10.1002/bit.25561
Ho SCL, Muriel B, Feng H, Mariati, Song Z, Yap MGS, Yang Y (2012) IRES-mediated tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol 157:130–139. doi:10.1016/j.jbiotec.2011.09.023
Hoch H, Chanutin A (1954) Albumin from heated human plasma. I. Preparation and electrophoretic properties. Arch Biochem Biophys 51:271–276
Lide DR (1990) CRC handbook of chemistry and physics (70th Ed.). CRC Press, Boca Raton (FL)
Morais V, Massaldi H (2012) A model mechanism for protein precipitation by caprylic acid: application to plasma purification. Biotechnol Appl Biochem 59:50–54. doi:10.1002/bab.68
Nian R, Chuah C, Lee J, Gan HT, Latiff SM, Lee WY, Vagenende V, Yang YS, Gagnon P (2013) Void exclusion of antibodies by grafted-ligand porous particle anion exchangers. J Chromatogr A 1282:127–132. doi:10.1016/j.chroma.2013.01.065
Nian R, Zhang W, Tan L, Lee J, Bi X, Yang Y, Gan HT, Gagnon P (2015) Advance chromatin extraction improves capture performance of protein a affinity chromatography. J Chromatogr A 1431:1–7. doi:10.1016/j.chroma.2015.12.044
Open drug and drug target database. (2005) http://www.drugbank.ca/drugs/DB00072
Russo C, Callegaro L, Lanza E, Ferrone S (1983) Purification of IgG monoclonal antibody by caprylic acid precipitation. J Immunol Methods 65:269–271. doi:10.1016/0022-1759(83)90324-1
Schlatter S, Stansfield SH, Dinnis DM, Racher AJ, Birch JR, James DC (2005) On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells. Biotechnol Prog 21:122–133. doi:10.1021/bp049780w
Singh N, Arunkumar A, Chollangi S, Tan Z, Borys M, Zheng JL (2016) Clarification technologies for monoclonal antibody manufacturing processes: current state and future perspectives. Biotechnol Bioeng 113:698–716. doi:10.1002/bit.25810
Sneekes EJ, Han J, Elliot M, Ausio J, Swart R, Heck AJR, Borchers C (2009) Accurate molecular weight analysis of histones using FFE and RP-HPLC on monolithic capillary columns. J Sep Sci 32:2691–2698. doi:10.1002/jssc.200800627
Steinbuch M, Audran R (1969) The isolation of IgG from mammalian sera with the aid of caprylic acid. Arch Biochem Biophys 134:279–284. doi:10.1016/0003-9861(69)90285-9
Temponi M, Kageshita T, Perosa F, Ono R, Okada H, Ferrone S (1989) Purification of murine IgG monoclonal antibodies by precipitation with caprylic acid: comparison with other methods of purification. Hybridoma 8:85–95. doi:10.1089/hyb.1989.8.85
Zheng J, Wang L, Twarowska B, Laino S, Sparks C, Smith T, Russell R, Wang M (2015) Caprylic acid-induced impurity precipitation from protein a capture column elution pool to enable a two chromatography step process for monoclonal antibody purification. Biotechnol Prog 259:1515–1525. doi:10.1002/btpr.2154
This work was financially supported by QIBEBT (Qingdao Institute of Bioenergy and Bioprocess Technology) Start-up Fund (No. Y571061905) and also by the Biomedical Research Council of A*STAR and Exploit Technologies Pte Ltd., of Singapore (No. ETPL/12-R15GAP-0009).
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
About this article
Cite this article
Chen, Q., Toh, P., Sun, Y. et al. Histone-dependent IgG conservation in octanoic acid precipitation and its mechanism. Appl Microbiol Biotechnol 100, 9933–9941 (2016). https://doi.org/10.1007/s00253-016-7719-x
- Octanoic acid
- Light chain