In order to retain structural and functional integrity, protein medicines are frequently stabilized with excipients in aqueous solutions. The goal of this investigation was to see how stable IL-2 is with excipients that are acceptable for cell therapy. We investigated the time-dependent stability of commercially available recombinant IL-2 in aqueous solutions (CTS, RPMI, PBS, and water) at different temperatures [2°C - 8°C, room temperature (20°C ± 2°C) and 37°C] in the presence of excipients (EDTA, methionine, histidine, and glycine) over a period of up to 30 days. To detect and quantify IL-2, reversed phase high performance liquid chromatography was employed. Electrophoresis on a sodium dodecyl sulfate polyacrylamide gel was used to assess conformational stability. We discovered that IL-2 stability was improved in aqueous solutions including excipients, and that it may have retained its biological activity and sterility in these conditions.
References
[1]
Morgan, D.A., Ruscetti, F.W. and Gallo, R. (1976) Selective in Vitro Growth of T Lymphocytes from Normal Human Bone Marrows. Science, 193, 1007-1008. https://doi.org/10.1126/science.181845
[2]
Rosenberg, S.A., Yang, J.C., White, D.E. and Steinberg, S.M. (1998) Durability of Complete Responses in Patients with Metastatic Cancer Treated with High-Dose Interleukin-2: Identification of the Antigens Mediating Response. Annals of Surgery, 228, 307-319.
[3]
Taniguchi, T., Matsui, H., Fujita, T., Takaoka, C., Kashima, N., Yoshimoto, R. and Hamuro, J. (1983) Structure and Expression of a Cloned cDNA for Human Interleukin-2. Nature, 302, 305-310. https://doi.org/10.1038/302305a0
Mark, D.F., Lin, L.S. and Lu, S.Y. (1985) Human Recombinant Interleukin-2 Muteins. United States Patent 4,518,584, May 21.
[6]
Hora, M.S., Rana, R.K., Wilcox, C.L., Katre, N.V., Hirtzer, P., Wolfe, S.N. and Thomson, J.W. (1992) Development of a Lyophilized Formulation of Interleukin-2. Developments in Biological Standardization, 74, 295-303.
[7]
Vemuri, S. (1992) Lyophilization Cycle Development for Interleukin-2. Developments in Biological Standardization, 74, 341-351.
[8]
Carpenter, J.F., Pikal, M.J., Chang, B.S. and Randolph, T.W. (1997) Rational Design of Stable Lyophilized Protein Formulations: Some Practical Advice. Pharmaceutical Research, 14, 969-975. https://doi.org/10.1023/A:1012180707283
[9]
Cadée, J.A., van Steenbergen, M.J., Versluis, C., Heck, A.J., Underberg, W.J., den Otter, W., Jiskoot, W. and Hennink, W.E. (2001) Oxidation of Recombinant Human Interleukin-2 by Potassium Peroxodisulfate. Pharmaceutical Research, 18, 1461-1467. https://doi.org/10.1023/A:1012213108319
[10]
Reyes, N., Ruiz, L., Aroche, K., Gerónimo, H., Brito, O. and Hardy, E. (2005) Stability of Ala 125 Recombinant Human Interleukin-2 in Solution. Journal of Pharmacy and Pharmacology, 57, 31-37. https://doi.org/10.1211/0022357055182
[11]
Gounili, P.K. (1999) The Effect of pH and Temperature on the Conformational Stability of Recombinant Human Interleukin-2. Doctoral Dissertations, AAI9942575. https://opencommons.uconn.edu/dissertations/AAI9942575
[12]
Vlasveld, L.T., Beijnen, J.H., Sein, J.J., Rankin, E.M., Melief, C.J. and Hekman, A. (1993) Reconstitution of Recombinant Interleukin-2 (rIL-2): A Comparative Study of Various rIL-2 Muteins. European Journal of Cancer, 29, 1977-1979. https://doi.org/10.1016/0959-8049(93)90456-P
[13]
Wang, W. (1999) Instability, Stabilization, and Formulation of Liquid Protein Pharmaceuticals. International Journal of Pharmaceutics, 185, 129-188. https://doi.org/10.1016/S0378-5173(99)00152-0
[14]
Kunitani, M., Hirtzer, P., Johnson, D., Halenbeck, R., Boosman, A. and Koths, K. (1986) Reversed-Phase Chromatography of Interleukin-2 Muteins. Journal of Chromatography A, 359, 391-402. https://doi.org/10.1016/0021-9673(86)80093-0
[15]
Duncan, M. (1995) Influence of Surfactants upon Protein/Peptide Adsorption to Glass and Polypropylene. International Journal of Pharmaceutics, 120, 179-188. https://doi.org/10.1016/0378-5173(94)00402-Q
[16]
Medvec Medvec, A.R., Ecker, C., Kong, H., Winters, E.A., Glover, J., Varela-Rohena, A. and Riley, J.L. (2017) Improved Expansion and in Vivo Function of Patient T Cells by a Serum-free Medium. Molecular Therapy—Methods & Clinical Development, 8, 65-74. https://doi.org/10.1016/j.omtm.2017.11.001
[17]
Wei, T., Kaewtathip, S. and Shing, K. (2009) Effect on Protein Adsorption at Liquid/Solid Interface. The Journal of Physical Chemistry, 113, 2053-2062. https://doi.org/10.1021/jp806586n
[18]
Prakasha Gowda, A.S., Schaefer, A.D. and Schuck, T.K. (2020) A Simple RP-HPLC Method for the Stability-Indicating Determination of Nacetyl-L-cysteine and N,N’-diacetyl-L-cystine in Cell Culture Media. Cell & Gene Therapy Insights, 6, 303-323. https://doi.org/10.18609/cgti.2020.041
[19]
Laemmli, U.K. (1970) Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 227, 680-685. https://doi.org/10.1038/227680a0
[20]
Johnston, T.P. (1996) Adsorption of Recombinant Human Granulocyte Colony Stimulating Factor (rhG-CSF) to Polyvinyl Chloride, Polypropylene, and Glass: Effect of Solvent Additives. PDA Journal of Pharmaceutical Science and Technology, 50, 238-245.
[21]
Tzannis, S.T., Hrushesky, W.J.M., Wood, P.A. and Przybycien, T.M. (1997) Adsorption of a Formulated Protein on a Drug Delivery Device Surface. Journal of Colloid and Interface Science, 189, 216-228. https://doi.org/10.1006/jcis.1997.4841
[22]
Andersson, M.M., Breccia, J.D. and Hatti-Kaul, R. (2000) Stabilizing Effect of Chemical Additives against Oxidation of Lactate Dehydrogenase. Biotechnology and Applied Biochemistry, 32, 145-153. https://doi.org/10.1042/BA20000014
[23]
Moya, G., González, L.J., Huerta, V., García, Y., Morera, V., Pérez, D., Breña, F. and Arana, M. (2002) Isolation and Characterization of Modified Species of a Mutated (Cys125-Ala) Recombinant Human Interleukin-2. Journal of Chromatography A, 971, 129-142. https://doi.org/10.1016/S0021-9673(02)00845-2
[24]
Levine, R.L., Mosoni, L., Berlett, B.S. and Stadtman, E.R. (1996) Methionine Residues as Endogenous Antioxidants in Proteins. Proceedings of the National Academy of Sciences of the United States of America, 93, 15036-15040. https://doi.org/10.1073/pnas.93.26.15036
[25]
Sebeka, H.K., Starkuviene, B., Trepsiene, O.V., Pauliukonis, A.A. and Bumelis, V.A. (2001) Comparative Effects of Stabilizing Additives on the Rates of Heat Inactivation of Recombinant Human Interferon Alpha-2b in Solution. Antiviral Research, 50, 117-127. https://doi.org/10.1016/S0166-3542(01)00131-0
[26]
Chen, B.L., Arakawa, T., Hsu, E., Narhi, L.O., Tressel, T.J. and Chien, S.L. (1994) Strategies to Suppress Aggregation of Recombinant Keratinocyte Growth Factor during Liquid Formulation Development. Journal of Pharmaceutical Sciences, 83, 1657-1661. https://doi.org/10.1002/jps.2600831204
[27]
Choi, N.S., Hahm, J.H., Maeng, P.J. and Kim, S.H. (2005) Comparative Study of Enzyme Activity and Stability of Bovine and Human Plasmins in Electrophoretic Reagents, Beta-Mercaptoethanol, DTT, SDS, Triton X-100, and Urea. Journal of Biochemistry and Molecular Biology, 38, 177-181. https://doi.org/10.5483/BMBRep.2005.38.2.177
[28]
Donbrow, M., Hamburger, R. and Azaz, E. (1975) Surface Tension and Cloud Point Changes of Polyoxyethylenic Non-Ionic Surfactants during Autoxidation. Journal of Pharmacy and Pharmacology, 27, 160-166. https://doi.org/10.1111/j.2042-7158.1975.tb09430.x
[29]
Koley, D. and Bard, A.J. (2010) Triton X-100 Concentration Effects on Membrane Permeability of a Single HeLa Cell by Scanning Electrochemical Microscopy (SECM). Proceedings of the National Academy of Sciences of the United States of America, 107, 16783-16787. https://doi.org/10.1073/pnas.1011614107
[30]
Cromwell, M.E., Hilario, E. and Jacobson, F. (2006) Protein Aggregation and Bioprocessing. The AAPS Journal, 8, E572-E579. https://doi.org/10.1208/aapsj080366
[31]
Grabski, A.C. (2009) Advances in Preparation of Biological Extracts for Protein Purification. Methods in Enzymology, 463, 285-303. https://doi.org/10.1016/S0076-6879(09)63018-4
[32]
Ellman, G.L. (1959) Tissue Sulfhydryl Groups. Archives of Biochemistry and Biophysics, 82, 70-77. https://doi.org/10.1016/0003-9861(59)90090-6
[33]
Chang, S.H., Teshima, G.M., Milby, T., Gillece-Castro, B. and Canova-Davis, E. (1997) Metal-Catalyzed Photooxidation of Histidine in Human Growth Hormone. Analytical Biochemistry, 244, 221-227. https://doi.org/10.1006/abio.1996.9899
[34]
Jones, C. (1981) Sulfur in Proteins: by Y M Torchinsky (Translated by W Wittenberg). pp 294. Pergamon Press, Oxford. (Original in Russian, 1977). $96 ISBN 0-08-023778-9. Biochemical Education, 10, 123. https://www.sciencedirect.com/science/article/pii/030744128290108X https://doi.org/10.1016/0307-4412(82)90108-X
[35]
Pikal-Cleland, K.A., Cleland, J.L., Anchordoquy, T.J. and Carpenter, J.F. (2002) Effect of Glycine on pH Changes and Protein Stability during Freeze-Thawing in Phosphate Buffer Systems. Journal of Pharmaceutical Sciences, 91, 1969-1979. https://doi.org/10.1002/jps.10184
[36]
Chen, B., Bautista, R., Yu, K., Zapata, G.A., Mulkerrin, M.G. and Chamow, S.M. (2003) Influence of Histidine on the Stability and Physical Properties of a Fully Human Antibody in Aqueous and Solid Forms. Pharmaceutical Research, 20, 1952-1960. https://doi.org/10.1023/B:PHAM.0000008042.15988.c0
[37]
Falconer, R.J., Chan, C., Hughes, K. and Munro, T.P. (2011) Stabilization of a Monoclonal Antibody during Purification and Formulation by Addition of Basic Amino Acid Excipients. Journal of Chemical Technology & Biotechnology, 86, 942-948. https://doi.org/10.1002/jctb.2657
