Two-Stage Purification of Recombinant Antibodies Using Non-Ionic Detergent Prevents Endotoxin-Induced Macrophage Activation: The Case of Bispecific Mini-Antibody MYSTI-2

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Yuliya A. Khodak^1,2, Anna M. Litvinova^1,3, Fedor A. Sysonov^1,4, Marina S. Drutskaya^1,3,4, Irina V. Safenkova⁵, Sergei A. Nedospasov^1,3,4, and Ivan I. Vorobiev^2,a*

¹Laboratory of Molecular Mechanisms of Immunity, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia

²Laboratory of Mammalian Cell Bioengineering, Research Center of Biotechnology, Skryabin Institute of Bioengineering, Russian Academy of Sciences, 119071 Moscow, Russia

³Immunology and Biomedicine Program, Center for Genetics and Life Sciences, Sirius University of Science and Technology, 354340 Sochi, Krasnodar Krai, Russia

⁴Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

⁵Laboratory of Immunobiochemistry, Research Center of Biotechnology, Bach Institute of Biochemistry, Russian Academy of Sciences, 119071 Moscow, Russia

^* To whom correspondence should be addressed.

Received: January 28, 2026; Revised: March 2, 2026; Accepted: March 6, 2026

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Escherichia coli is one of the most common producers of recombinant proteins, including therapeutic antibody fragments. However, the outer membrane of E. coli contains high levels of lipopolysaccharide (LPS, also known as endotoxin), which can activate innate immune receptors, trigger immune responses, and induce systemic inflammation that may progress to septic shock. Ensuring extremely low endotoxin levels in preparations intended for in vivo applications is critically important. In this study, we investigated the endotoxin content in preparations of the bispecific mini-antibody MYSTI-2 produced in two E. coli strains: the Rosetta strain, which synthesizes conventional LPS, and the ClearColi strain, which synthesizes potentially non-toxic form of LPS. Our results demonstrate that near-complete removal of LPS can be achieved only through the use of a non-ionic detergent during purification, regardless of the bacterial strain used for protein production.

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KEY WORDS: recombinant proteins, endotoxin, bispecific antibodies

DOI: 10.1134/S0006297926600225

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