A Manufacturing Strategy Utilizing a Continuous-Mode Reactor toward Homogeneous PEGylated Bioconjugate Production^[1]

Abstract

Protein PEGylation is a traditional bioconjugation technology that enhances the therapeutic efficacy and in vivo half-life of proteins by the formation of covalent bonds with highly activated ester group linked polyethylene glycol (PEG). However, the high reactivity of these reagents induces a random reaction with lysine residues on the protein surface, resulting in a heterogeneous mixture of PEGylated proteins. Moreover, the traditional batch-mode reaction has risks relating to scalability and aggregation. To overcome these risks of traditional batch-mode PEGylation, a manufacturing strategy utilizing structural analysis and a continuous-flow-mode reaction was examined. A solvent exposure analysis revealed the most reactive lysine of a protein, and the continuous-flow mode modified this lysine to achieve the mono-PEGylation of two different proteins within 2 seconds. This ultrarapid modification reaction can be applied to the gram-scale manufacturing of PEGylated bioconjugates without generating aggregates. A similar trend of the exposure level of protein lysine and mono-selectivity performed by continuous-flow PEGylation was observed, which indicated that this manufacturing strategy has the potential to be applied to the production of a wide variety of bioconjugates.

Key words

continuous-flow synthesis - microreactor - PEGylation - cytokines - bioconjugates

Publication History

Received: 22 February 2023

Accepted after revision: 19 April 2023

Accepted Manuscript online:
19 April 2023

Article published online:
30 May 2023

• References

• 1 The submitted (pre-acceptance) version of this manuscript was published as a preprint in ChemRxiv: Nakahara Y, Endo Y, Takahashi K, Kawaguchi T, Kato K, Matsuda Y, Nagaki A. ChemRxiv 2023; preprint
• 2 Current address: Ajinomoto Bio-Pharma Services, 11040 Roselle Street, San Diego, CA 92121, USA.
• 3a Turecek PL, Bossard MJ, Schoetens F, Ivens IA. J. Pharm. Sci. 2016; 105: 460
• 3b Mishra P, Nayak B, Dey RK. Asian J. Pharm. Sci. 2016; 11: 337
• 3c Mao L, Russell AJ, Carmali S. Bioconjugate Chem. 2022; 33: 1643
• 4 Armengol ES, Unterweger A, Laffleur F. Drug Dev. Ind. Pharm. 2022; 48: 129
• 5 Rajan R, Ahmed S, Sharma N, Kumar N, Debas A, Matsumura K. Mater. Adv. 2021; 2: 1139
• 6 Rossetti I, Compagnoni M. Chem. Eng. J. 2016; 296: 56
• 7 Baumann M, Moody TS, Smyth M, Wharry S. Org. Process Res. Dev. 2020; 24: 1802
• 8 Hughes DL. Org. Process Res. Dev. 2020; 24: 1850
• 9 Takahashi Y, Nagaki A. Molecules 2019; 24: 1532
• 10 Movsisyan M, Delbeke EI, Berton JK, Battilocchio C, Ley SV, Stevens CV. Chem. Soc. Rev. 2016; 45: 4892
• 11 Nakahara Y, Mendelsohn BA, Matsuda Y. Org. Process Res. Dev. 2022; 26: 2766
• 12 Ingold O, Pfister D, Morbidelli M. React. Chem. Eng. 2016; 1: 218
• 13 Shang X, Ghosh R. J. Membr. Sci. 2014; 451: 177
• 14 Madadkar P, Selvaganapathy PR, Ghosh R. Biomicrofluidics 2018; 12: 044114
• 15 Kateja N, Nitika, Dureja S, Rathore AS. J. Biotechnol. 2020; 322: 79
• 16 Matsuda Y, Leung M, Tawfiq Z, Fujii T, Mendelsohn BA. Anal. Sci. 2021; 37: 1171
• 17 Yamazaki S, Shikida N, Takahashi K, Matsuda Y, Inoue K, Shimbo K, Mihara Y. Bioorg. Med. Chem. Lett. 2021; 51: 128360
• 18 Matsuda Y, Chakrabarti A, Takahashi K, Yamada K, Nakata K, Okuzumi T, Mendelsohn BA. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2021; 1177: 122753
• 19 Tsutsumi YE, Tsunoda SS, Kamada H, Kihira T, Kaneda Y, Ohsugi Y, Mayumi T. Thromb. Haemost. 1997; 77: 168
• 20 Tsunoda S, Ishikawa T, Watanabe M, Kamada H, Yamamoto Y, Tsutsumi Y, Hirano T, Mayumi T. Br. J. Haematol. 2001; 112: 181
• 21 Somers W, Stahl M, Seehra JS. EMBO J. 1997; 16: 989
• 22 Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Žídek A, Potapenko A, Bridgland A, Meyer C, Kohl SA. A, Ballard AJ, Cowie A, Romera-Paredes B, Nikolov S, Jain R, Adler J, Back T, Petersen S, Reiman D, Clancy E, Zielinski M, Steinegger M, Pacholska M, Berghammer T, Bodenstein S, Silver D, Vinyals O, Senior AW, Kavukcuoglu K, Kohli P, Hassabis D. Nature 2021; 596: 583
• 23 Varadi M, Anyango S, Deshpande M, Nair S, Natassia C, Yordanova G, Yuan D, Stroe O, Wood G, Laydon A, Žídek A, Green T, Tunyasuvunakool K, Petersen S, Jumper J, Clancy E, Green R, Vora A, Lutfi M, Figurnov M, Cowie A, Hobbs N, Kohli P, Kleywegt G, Birney E, Hassabis D, Velankar S. Nucleic Acids Res. 2022; 50: D439
• 24 Konermann L. J. Phys. Chem. A 1999; 103: 7210
• 25 NOF Corporation (accessed May 8, 2023). https://www.nofamerica.com/store/index.php?dispatch=categories.view&category_id=7
• 26 Endo Y, Furusawa M, Shimazaki T, Takahashi Y, Nakahara Y, Nagaki A. Org. Process Res. Dev. 2019; 23: 635
• 27 Taylor CJ, Baker A, Chapman MR, Reynolds WR, Jolley KE, Clemens G, Smith GE, Blacker AJ, Chamberlain TW, Christie SD. R, Taylor BA, Bourne RA. J. Flow Chem. 2021; 11: 75
• 28 Reckamp JM, Bindels A, Duffield S, Liu YC, Bradford E, Ricci E, Susanne F, Rutter A. Org. Process Res. Dev. 2017; 21: 816
• 29 Ehrfeld W, Golbig K, Hessel V, Löwe H, Richter T. Ind. Eng. Chem. Res. 1999; 38: 1075
• 30 Fujii T, Reiling C, Quinn C, Kliman M, Mendelsohn BA, Matsuda Y. Explor. Targeted Anti-Tumor Ther. 2021; 2: 576
• 31 Tawfiq Z, Matsuda Y, Alfonso MJ, Clancy C, Robles V, Leung M, Mendelsohn BA. Anal. Sci. 2020; 36: 871
• 32 Savojardo C, Manfredi M, Martelli PL, Casadio R. Front. Mol. Biosci. 2021; 7: 626363
• 33 Kajander T, Kahn PC, Passila SH, Cohen DC, Lehtiö L, Adolfsen W, Warwicker J, Schell U, Goldman A. Structure 2000; 8: 1203
• 34 An FMR has the advantage of high throughput to find appropriate reaction conditions. As a hypothetical case study, we have attempted to compare the estimated development time for both batch and FMR cases. In the case of batch development, it is assumed that each parameter study takes 10 minutes to find ideal scale-up conditions, so a total of 300 minutes is required if 30 parameter studies are performed. On the other hand, in the case of FMR development, the run time for each parameter study is less than 1 minute. Therefore, a total of only 30 minutes is required to complete 30 parameter studies.
• 35 Fujii T, Matsuda Y, Seki T, Shikida N, Iwai Y, Ooba Y, Takahashi K, Isokawa M, Kawaguchi S, Hatada N, Watanabe T, Takasugi R, Nakayama A, Shimbo K, Mendelsohn BA, Okuzumi T, Yamada K. Bioconjugate Chem. 2023; 34: 728
• 36 Ejima D, Watanabe M, Sato Y, Date M, Yamada N, Takahara Y. Biotechnol. Bioeng. 1999; 62: 301
• 37 Weiss MS, Palm GJ, Hilgenfeld R. Acta Crystallogr., Sect. D 2000; 56: 952
• 38 Matsuda Y, Seki T, Yamada K, Ooba Y, Takahashi K, Fujii T, Kawaguchi S, Narita T, Nakayama A, Kitahara Y, Mendelsohn BA, Okuzumi T. Mol. Pharmaceutics 2021; 18: 4058
• 39 Matsuda Y, Tawfiq Z, Leung M, Mendelsohn BA. ChemistrySelect 2020; 5: 8435
• 40 Seki T, Yamada K, Ooba Y, Fujii T, Narita T, Nakayama A, Kitahara Y, Mendelsohn BA, Matsuda Y, Okuzumi T. Front. Biosci.-Landmark 2022; 27: 234