Strategies to stabilize compact folding and minimize aggregation of antibody-based fragments


Monoclonal antibodies (mAbs) have proven to be useful for development of new therapeutic drugs and diagnostic techniques. To overcome the difficulties posed by their complex structure and folding, reduce undesired immunogenicity, and improve pharmacoki- netic properties, a plethora of different Ab fragments have been developed. These include recombinant Fab and Fv segments that can display improved properties over those of the original mAbs upon which they are based. Antibody (Ab) fragments such as Fabs, scFvs, diabodies, and nanobodies, all contain the variable Ig domains responsible for binding to specific antigenic epitopes, allowing for specific targeting of pathological cells and/or molecules. These fragments can be easier to produce, purify and refold than a full Ab, and due to their smaller size they can be well absorbed and distributed into target tissues. However, the physicochemical and structural properties of the immunoglobulin (Ig) domain, upon which the folding and conformation of all these Ab fragments is based, can limit the stability of Ab-based drugs. The Ig domain is fairly sensitive to unfolding and aggregation when produced out of the structural context of an intact Ab molecule. When unfolded, Ab fragments may lose their specificity as well as establish non-native interactions leading to protein aggregation. Aggregated antibody fragments display altered pharmacokinetic and immunogenic properties that can augment their toxicity. Therefore, much effort has been placed in understanding the factors impacting the stability of Ig folding at two different levels: 1) intrinsically, by studying the effects of the amino acid sequence on Ig folding; 2) extrinsically, by determining the environmental conditions that may influence the stability of Ig folding. In this review we will describe the structure of the Ig domain, and the factors that impact its stability, to set the context for the different approaches currently used to achieve stable recombinant Ig domains when pursuing the development of Ab fragment-based biotechnologies.

Share and Cite:

Gil, D. and Schrum, A. (2013) Strategies to stabilize compact folding and minimize aggregation of antibody-based fragments. Advances in Bioscience and Biotechnology, 4, 73-84. doi: 10.4236/abb.2013.44A011.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Cambier, J.C. and Campbell, K.S. (1992) Membrane immunoglobulin and its accomplices: New lessons from an old receptor. The FASEB Journal, 6, 3207-3217.
[2] Schroeder Jr., H.W. and Cavacini, L. (2010) Structure and function of immunoglobulins. Journal of Allergy and Clinical Immunology, 125, S41-S52. doi:10.1016/j.jaci.2009.09.046
[3] Schroeder Jr., H.W., Mortari, F., Shiokawa, S., Kirkham, P.M., Elgavish, R.A. and Bertrand 3rd, F.E. (1995) Developmental regulation of the human antibody repertoire. Annals of the New York Academy of Sciences, 764, 242- 260. doi:10.1111/j.1749-6632.1995.tb55834.x
[4] Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256, 495-497. doi:10.1038/256495a0
[5] Hudson, P.J. and Souriau, C. (2003) Engineered antibodies. Nature Medicine, 9, 129-134. doi:10.1038/nm0103-129
[6] Bumbaca, D., Boswell, C.A., Fielder, P.J. and Khawli, L.A. (2012) Physiochemical and biochemical factors influencing the pharmacokinetics of antibody therapeutics. AAPS Journals, 14, 554-558. doi:10.1208/s12248-012-9369-y
[7] Olafsen, T. (2012) Fc engineering: Serum half-life modulation through FcRn binding. Methods in Molecular Biology, 907, 537-556.
[8] Chatenoud, L. and Bluestone, J.A. (2007) CD3-specific antibodies: A portal to the treatment of autoimmunity. Nature Reviews Immunology, 7, 622-632. doi:10.1038/nri2134
[9] Chatenoud, L. (2003) CD3-specific antibody-induced active tolerance: From bench to bedside. Nature Reviews Immunology, 3, 123-132. doi:10.1038/nri1000
[10] Wu, A.M. and Senter, P.D. (2005) Arming antibodies: Prospects and challenges for immunoconjugates. Nature Biotechnology, 23, 1137-1146. doi:10.1038/nbt1141
[11] Goldenberg, D.M. and Sharkey, R.M. (2007) Novel radiolabeled antibody conjugates. Oncogene, 26, 3734-3744. doi:10.1038/sj.onc.1210373
[12] Kenanova, V. and Wu, A.M. (2006) Tailoring antibodies for radionuclide delivery. Expert Opinion on Drug Delivery, 3, 53-70. doi:10.1517/17425247.3.1.53
[13] Holliger, P. and Hudson, P.J. (2005) Engineered antibody fragments and the rise of single domains. Nature Biotechnology, 23, 1126-1136. doi:10.1038/nbt1142
[14] Perchiacca, J.M. and Tessier, P.M. (2012) Engineering aggregation-resistant antibodies. Annual Review of Chemical and Biomolecular Engineering, 3, 263-286. doi:10.1146/annurev-chembioeng-062011-081052
[15] Daugherty, A.L. and Mrsny, R.J. (2006) Formulation and delivery issues for monoclonal antibody therapeutics. Advanced Drug Delivery Reviews, 58, 686-706. doi:10.1016/j.addr.2006.03.011
[16] Bork, P., Holm, L. and Sander, C. (1994) The immunoglobulin fold. Structural classification, sequence patterns and common core. Journal of Molecular Biology, 242, 309- 320. doi:10.1016/S0022-2836(84)71582-8
[17] Halaby, D.M., Poupon, A. and Mornon, J. (1999) The immunoglobulin fold family: Sequence analysis and 3D structure comparisons. Protein Engineering, 12, 563-571. doi:10.1093/protein/12.7.563
[18] Williams, A.F. and Barclay, A.N. (1988) The immunoglobulin superfamily—Domains for cell surface recognition. Annual Review of Immunology, 6, 381-405. doi:10.1146/annurev.iy.06.040188.002121
[19] Rothlisberger, D., Honegger, A. and Pluckthun, A. (2005) Domain interactions in the Fab fragment: A comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability. Journal of Molecular Biology, 347, 773-789. doi:10.1016/j.jmb.2005.01.053
[20] Nieba, L., Honegger, A., Krebber, C. and Pluckthun, A. (1997) Disrupting the hydrophobic patches at the antibody variable/constant domain interface: Improved in vivo folding and physical characterization of an engineered scFv fragment. Protein Engineering, 10, 435-444. doi:10.1093/protein/10.4.435
[21] Sutton, B.J. and Phillips, D.C. (1983) The three-dimensional structure of the carbohydrate within the Fc fragment of immunoglobulin G. Biochemical Society Transactions, 11, 130-132.
[22] Wang, W., Singh, S., Zeng, D.L., King, K. and Nema, S. (2007) Antibody structure, instability, and formulation. Journal of Pharmaceutical Sciences, 96, 1-26. doi:10.1002/jps.20727
[23] Krapp, S., Mimura, Y., Jefferis, R., Huber, R. and Sondermann, P. (2003) Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity. Journal of Molecular Biology, 325, 979-989. doi:10.1016/S0022-2836(02)01250-0
[24] Kumar, R. (2009) Role of naturally occurring osmolytes in protein folding and stability. Archives of Biochemistry and Biophysics, 491, 1-6. doi:10.1016/
[25] Jager, M. and Pluckthun, A. (1999) Domain interactions in antibody Fv and scFv fragments: Effects on unfolding kinetics and equilibria. FEBS Letters, 462, 307-312. doi:10.1016/S0014-5793(99)01532-X
[26] Worn, A. and Pluckthun, A. (2001) Stability engineering of antibody single-chain Fv fragments. Journal of Molecular Biology, 305, 989-1010. doi:10.1006/jmbi.2000.4265
[27] Shimba, N., Torigoe, H., Takahashi, H., Masuda, K., Shimada, I., Arata, Y. and Sarai, A. (1995) Comparative thermodynamic analyses of the Fv, Fab* and Fab fragments of anti-dansyl mouse monoclonal antibody. FEBS Letters, 360, 247-250. doi:10.1016/0014-5793(95)00113-N
[28] Glockshuber, R., Malia, M., Pfitzinger, I. and Pluckthun, A. (1990) A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry, 29, 1362-1367. doi:10.1021/bi00458a002
[29] Brinkmann, U., Reiter, Y., Jung, S.H., Lee, B. and Pastan, I. (1993) A recombinant immunotoxin containing a disulfide-stabilized Fv fragment. Proceedings of the National Academy of Sciences of the United States of America, 90, 7538-7542. doi:10.1073/pnas.90.16.7538
[30] Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S., Kaufman, B.M., Lee, S.M., Lee, T., Pope, S.H., Riordan, G.S. and Whitlow, M. (1988) Single-chain antigen-binding proteins. Science, 242, 423-426. doi:10.1126/science.3140379
[31] Huston, J.S., Levinson, D., Mudgett-Hunter, M., Tai, M.S., Novotny, J., Margolies, M.N., Ridge, R.J., Bruccoleri, R.E., Haber, E., Crea, R., et al. (1988) Protein engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 85, 5879-5883. doi:10.1073/pnas.85.16.5879
[32] Young, N.M., MacKenzie, C.R., Narang, S.A., Oomen, R.P. and Baenziger, J.E. (1995) Thermal stabilization of a single-chain Fv antibody fragment by introduction of a disulphide bond. FEBS Letters, 377, 135-139. doi:10.1016/0014-5793(95)01325-3
[33] Ewert, S., Honegger, A. and Pluckthun, A. (2004) Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering. Methods, 34, 184-199. doi:10.1016/j.ymeth.2004.04.007
[34] Bennett, M.J. and Eisenberg, D. (2004) The evolving role of 3D domain swapping in proteins. Structure, 12, 339- 1341. doi:10.1016/j.str.2004.07.004
[35] Gronenborn, A.M. (2009) Protein acrobatics in pairsdimerization via domain swapping. Current Opinion in Structural Biology, 19, 39-49. doi:10.1016/
[36] Rousseau, F., Schymkowitz, J. and Itzhaki, L.S. (2012) Implications of 3D domain swapping for protein folding, misfolding and function. Advances in Experimental Medicine and Biology, 747, 137-152. doi:10.1007/978-1-4614-3229-6_9
[37] Arndt, K.M., Muller, K.M. and Pluckthun, A. (1998) Factors influencing the dimer to monomer transition of an antibody single-chain Fv fragment. Biochemistry, 37, 12918-12926. doi:10.1021/bi9810407
[38] Holliger, P., Prospero, T. and Winter, G. (1993) Diabodies: Small bivalent and bispecific antibody fragments. Proceedings of the National Academy of Sciences of the United States of America, 90, 6444-6448. doi:10.1073/pnas.90.14.6444
[39] Kortt, A.A., Dolezal, O., Power, B.E. and Hudson, P.J. (2001) Dimeric and trimeric antibodies: High avidity scFvs for cancer targeting. Biomolecular Engineering, 18, 95-108. doi:10.1016/S1389-0344(01)00090-9
[40] Pluckthun, A. and Pack, P. (1997) New protein engineering approaches to multivalent and bispecific antibody fragments. Immunotechnology, 3, 83-105. doi:10.1016/S1380-2933(97)00067-5
[41] Marvin, J.S. and Zhu, Z. (2005) Recombinant approaches to IgG-like bispecific antibodies. Acta Pharmacologica Sinica, 26, 649-658. doi:10.1111/j.1745-7254.2005.00119.x
[42] De Marco, A. (2011) Biotechnological applications of recombinant single-domain antibody fragments. Microbial Cell Factories, 10, 44. doi:10.1186/1475-2859-10-44
[43] Dimitrov, D.S. (2009) Engineered CH2 domains (nanoantibodies). MAbs, 1, 26-28. doi:10.4161/mabs.1.1.7480
[44] Saerens, D., Ghassabeh, G.H. and Muyldermans, S. (2008) Single-domain antibodies as building blocks for novel therapeutics. Current Opinion in Pharmacology, 8, 600- 608. doi:10.1016/j.coph.2008.07.006
[45] Vincke, C. and Muyldermans, S. (2012) Introduction to heavy chain antibodies and derived Nanobodies. Methods in Molecular Biology, 911, 15-26.
[46] Borrebaeck, C.A. and Ohlin, M. (2002) Antibody evolution beyond nature. Nature Biotechnology, 20, 1189- 1190. doi:10.1038/nbt1202-1189
[47] Ward, E.S., Gussow, D., Griffiths, A.D., Jones, P.T. and Winter, G. (1989) Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature, 341, 544-546. doi:10.1038/341544a0
[48] Teillaud, J.L. (2012) From whole monoclonal antibodies to single domain antibodies: Think small. Methods in Molecular Biology, 911, 3-13.
[49] Wesolowski, J., Alzogaray, V., Reyelt, J., Unger, M., Juarez, K., Urrutia, M., Cauerhff, A., Danquah, W., Rissiek, B., Scheuplein, F., et al. (2009) Single domain antibodies: Promising experimental and therapeutic tools in infection and immunity. Medical Microbiology and Immunology, 198, 157-174. doi:10.1007/s00430-009-0116-7
[50] Harmsen, M.M. and De Haard, H.J. (2007) Properties, production, and applications of camelid single-domain antibody fragments. Applied Microbiology and Biotechnology, 77, 13-22. doi:10.1007/s00253-007-1142-2
[51] Perchiacca, J.M., Bhattacharya, M. and Tessier, P.M. (2011) Mutational analysis of domain antibodies reveals aggregation hotspots within and near the complementarity determining regions. Proteins, 79, 2637-2647. doi:10.1002/prot.23085
[52] Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hamers, C., Songa, E.B., Bendahman, N. and Hamers, R. (1993) Naturally occurring antibodies devoid of light chains. Nature, 363, 446-448. doi:10.1038/363446a0
[53] Muyldermans, S., Atarhouch, T., Saldanha, J., Barbosa, J.A. and Hamers, R. (1994) Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Engineering Design & Selection, 7, 1129-1135. doi:10.1093/protein/7.9.1129
[54] Barthelemy, P.A., Raab, H., Appleton, B.A., Bond, C.J., Wu, P., Wiesmann, C. and Sidhu, S.S. (2008) Comprehensive analysis of the factors contributing to the stability and solubility of autonomous human VH domains. The Journal of Biological Chemistry, 283, 3639-3654. doi:10.1074/jbc.M708536200
[55] Bond, C.J., Marsters, J.C. and Sidhu, S.S. (2003) Contributions of CDR3 to VHH domain stability and the design of monobody scaffolds for naive antibody libraries. Journal of Molecular Biology, 332, 643-655. doi:10.1016/S0022-2836(03)00967-7
[56] Jespers, L., Schon, O., James, L.C., Veprintsev, D. and Winter, G. (2004) Crystal structure of HEL4, a soluble, refoldable human V(H) single domain with a germ-line scaffold. Journal of Molecular Biology, 337, 893-903. doi:10.1016/j.jmb.2004.02.013
[57] Saerens, D., Pellis, M., Loris, R., Pardon, E., Dumoulin, M., Matagne, A., Wyns, L., Muyldermans, S. and Conrath, K. (2005) Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies. Journal of Molecular Biology, 352, 597-607. doi:10.1016/j.jmb.2005.07.038
[58] Vincke, C., Loris, R., Saerens, D., Martinez-Rodriguez, S., Muyldermans, S. and Conrath, K. (2009) General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. Journal of Molecular Biology, 284, 3273-3284. doi:10.1074/jbc.M806889200
[59] Desmyter, A., Transue, T.R., Ghahroudi, M.A., Thi, M.H., Poortmans, F., Hamers, R., Muyldermans, S. and Wyns, L. (1996) Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme. Nature Structural & Molecular Biology, 3, 803-811. doi:10.1038/nsb0996-803
[60] Rahbarizadeh, F., Ahmadvand, D. and Sharifzadeh, Z. (2011) Nanobody; an old concept and new vehicle for immunotargeting. Immunological Investigations, 40, 299- 338. doi:10.3109/08820139.2010.542228
[61] Coppieters, K., Dreier, T., Silence, K., De Haard, H., Lauwereys, M., Casteels, P., Beirnaert, E., Jonckheere, H., Van de Wiele, C., Staelens, L., et al. (2006) Formatted anti-tumor necrosis factor alpha VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis. Arthritis & Rheumatism, 54, 1856-1866. doi:10.1002/art.21827
[62] Cortez-Retamozo, V., Lauwereys, M., Hassanzadeh Gh, G., Gobert, M., Conrath, K., Muyldermans, S., De Baetselier, P. and Revets, H. (2002) Efficient tumor targeting by single-domain antibody fragments of camels. International Journal of Cancer, 98, 456-462. doi:10.1002/ijc.10212
[63] Muyldermans, S. (2001) Single domain camel antibodies: Current status. Journal of Biotechnology, 74, 277-302.
[64] Riechmann, L. and Muyldermans, S. (1999) Single domain antibodies: comparison of camel VH and camelised human VH domains. Journal of Immunological Methods, 231, 25-38. doi:10.1016/S0022-1759(99)00138-6
[65] Muyldermans, S., Cambillau, C. and Wyns, L. (2001) Recognition of antigens by single-domain antibody fragments: The superfluous luxury of paired domains. Trends in Biochemical Sciences, 26, 230-235. doi:10.1016/S0968-0004(01)01790-X
[66] Ewert, S., Cambillau, C., Conrath, K. and Pluckthun, A. (2002) Biophysical properties of camelid V(HH) domains compared to those of human V(H)3 domains. Biochemistry, 41, 3628-3636. doi:10.1021/bi011239a
[67] Nelson, A.D., Hoffmann, M.M., Parks, C.A., Dasari, S., Schrum, A.G. and Gil, D. (2012) IgG Fab fragments forming bivalent complexes by a conformational mechanism that is reversible by osmolytes. Journal of Biological Chemistry, 287, 42936-42950. doi:10.1074/jbc.M112.410217
[68] Kayser, V., Chennamsetty, N., Voynov, V., Forrer, K., Helk, B. and Trout, B.L. (2011) Glycosylation influences on the aggregation propensity of therapeutic monoclonal antibodies. Biotechnology Journal, 6, 38-44. doi:10.1002/biot.201000091
[69] Wu, S.J., Luo J, O’Neil, K.T., Kang. J., Lacy, E.R., Canziani, G., Baker, A., Huang, M., Tang, Q.M., Raju, T.S., et al. (2010) Structure-based engineering of a monoclonal antibody for improved solubility. Protein Engineering Design & Selection, 23, 643-651. doi:10.1093/protein/gzq037
[70] Miller, B.R., Demarest, S.J., Lugovskoy, A., Huang, F., Wu, X., Snyder, W.B., Croner, L.J., Wang, N., Amatucci, A., Michaelson, J.S. and Glaser, S.M. (2010) Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Engineering Design & Selection, 23, 549-557. doi:10.1093/protein/gzq028
[71] Perchiacca, J.M., Ladiwala, A.R., Bhattacharya, M. and Tessier, P.M. (2012) Aggregation-resistant domain antibodies engineered with charged mutations near the edges of the complementarity-determining regions. Protein Engineering Design & Selection, 25, 591-601. doi:10.1093/protein/gzs042
[72] Calarese, D.A., Scanlan, C.N., Zwick, M.B., Deechongkit, S., Mimura, Y., Kunert, R., Zhu, P., Wormald, M.R., Stanfield, R.L., Roux, K.H., et al. (2003) Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. Science, 300, 2065-2071. doi:10.1126/science.1083182
[73] Khan, S.H., Ahmad, N., Ahmad, F. and Kumar, R. (2010) Naturally occurring organic osmolytes: From cell physiology to disease prevention. IUBMB Life, 62, 891-895. doi:10.1002/iub.406
[74] Yancey, P.H. (2005) Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. The Journal of Experimental Biology, 208, 2819-2830. doi:10.1242/jeb.01730
[75] Yancey, P.H., Clark, M.E., Hand, S.C., Bowlus, R.D. and Somero, G.N. (1982) Living with water stress: Evolution of osmolyte systems. Science, 217, 1214-1222. doi:10.1126/science.7112124
[76] Arakawa, T., Tsumoto, K., Kita, Y., Chang, B. and Ejima, D. (2007) Biotechnology applications of amino acids in protein purification and formulations. Amino Acids, 33, 587-605. doi:10.1007/s00726-007-0506-3
[77] Bolen, D.W. (2004) Effects of naturally occurring osmolytes on protein stability and solubility: Issues important in protein crystallization. Methods, 34, 312-322. doi:10.1016/j.ymeth.2004.03.022
[78] Somero, G.N. (1986) Protons, osmolytes, and fitness of internal milieu for protein function. American Journal of Physiology, 251, R197-213.
[79] Bolen, D.W. and Baskakov, I.V. (2001) The osmophobic effect: natural selection of a thermodynamic force in protein folding. Journal of Molecular Biology, 310, 955-963. doi:10.1006/jmbi.2001.4819
[80] Street, T.O., Bolen, D.W. and Rose, G.D. (2006) A molecular mechanism for osmolyte-induced protein stability. Proceedings of the National Academy of Sciences of the United States of America, 103, 13997-14002. doi:10.1073/pnas.0606236103
[81] Arakawa, T. and Timasheff, S.N. (1985) The stabilization of proteins by osmolytes. Biophysical Journal, 47, 411-414. doi:10.1016/S0006-3495(85)83932-1
[82] Timasheff, S.N. (1992) Water as ligand: Preferential binding and exclusion of denaturants in protein unfolding. Biochemistry, 31, 9857-9864. doi:10.1021/bi00156a001
[83] Lee, J.C. (2000) Biopharmaceutical formulation. Current Opinion in Biotechnology, 11, 81-84. doi:10.1016/S0958-1669(99)00058-0
[84] Chi, E.Y., Krishnan, S., Randolph, T.W. and Carpenter, J.F. (2003) Physical stability of proteins in aqueous solution: Mechanism and driving forces in nonnative protein aggregation. Pharmaceutical Research, 20, 1325-1336. doi:10.1023/A:1025771421906
[85] Carpenter, J.F., Manning, M.C. and Randolph, T.W. (2002) Long-term storage of proteins. Current Protocols in Protein Science, Chapter 4, Unit 46.
[86] Barth, S., Huhn, M., Matthey, B., Klimka, A., Galinski, E.A. and Engert, A. (2000) Compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions. Applied and Environmental Microbiology, 66, 1572-1579. doi:10.1128/AEM.66.4.1572-1579.2000
[87] Samuel, D., Kumar, T.K., Ganesh, G., Jayaraman, G., Yang, P.W., Chang, M.M., Trivedi, V.D, Wang, S.L., Hwang, K.C., Chang, D.K. and Yu, C. (2000) Proline inhibits aggregation during protein refolding. Protein Science, 9, 344-352. doi:10.1110/ps.9.2.344
[88] 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. doi:10.1023/B:PHAM.0000008042.15988.c0
[89] Chen, B.L. and Arakawa, T. (1996) Stabilization of recombinant human keratinocyte growth factor by osmolytes and salts. Journal of Pharmaceutical Sciences, 85, 419-426. doi:10.1021/js9504393
[90] Arakawa, T., Dix, D.B. and Chang, B.S. (2003) The effects of protein stabilizers on aggregation induced by multiple-stresses. Yakugaku Zasshi, 123, 957-961. doi:10.1248/yakushi.123.957
[91] Tian, F., Sane, S. and Rytting, J.H. (2006) Calorimetric investigation of protein/amino acid interactions in the solid state. International Journal of Pharmaceutics, 310, 175-186. doi:10.1016/j.ijpharm.2005.12.009
[92] Zhang, M.Z., Wen, J., Arakawa, T. and Prestrelski, S.J. (1995) A new strategy for enhancing the stability of lyophilized protein: The effect of the reconstitution medium on keratinocyte growth factor. Pharmaceutical Research, 12, 1447-1452. doi:10.1023/A:1016219000963
[93] Zhang, M.Z., Pikal, K., Nguyen, T., Arakawa, T. and Prestrelski, S.J. (1996) The effect of the reconstitution medium on aggregation of lyophilized recombinant interleukin-2 and ribonuclease A. Pharmaceutical Research, 13, 643-646. doi:10.1023/A:1016074811306
[94] Ignatova, Z. and Gierasch, L.M. (2006) Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant. Proceedings of the National Academy of Sciences of the United States of America, 103, 13357- 13361. doi:10.1073/pnas.0603772103

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.