Optimization of Storage Stability of Lyophilized Actin Using Combinations of Disaccharides and Dextran  S.Dean Allison, Mark C. Manning, Theodore W. Randolph, Kim Middleton, Ashley Davis, John F. Carpenter  Journal of Pharmaceutical Sciences  Volume 89, Issue 2, Pages 199-214 (February 2000) DOI: 10.1002/(SICI)1520-6017(200002)89:2<199::AID-JPS7>3.0.CO;2-B Copyright © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association Terms and Conditions

Figure 1 Representative DSC thermograms of (A) sucrose and (B) trehalose formulations: (a) 1% sugar, (b) 1% sugar + 1% dextran, (c) 5% sugar, (d) 5% sugar + 1% dextran, (e) 1% dextran. Journal of Pharmaceutical Sciences 2000 89, 199-214DOI: (10.1002/(SICI)1520-6017(200002)89:2<199::AID-JPS7>3.0.CO;2-B) Copyright © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association Terms and Conditions

Figure 2 Area normalized, second‐derivative amide I spectra of actin dried with dextran; (dotted lines) native actin, (solid lines) spectra taken immediately after freeze‐drying, (dashed lines) spectra taken after 8 weeks at 45°C, (dot‐dashed lines) spectra taken after 8 weeks at 60°C. Bold solid lines represent the spectrum of actin dried without additives. Journal of Pharmaceutical Sciences 2000 89, 199-214DOI: (10.1002/(SICI)1520-6017(200002)89:2<199::AID-JPS7>3.0.CO;2-B) Copyright © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association Terms and Conditions

Figure 3 Circular dichroism spectra of rehydrated actin after drying with dextran: (dotted lines) native actin, (solid lines) actin rehydrated immediately after freeze‐drying, (dashed lines) actin rehydrated after 8 weeks at 45°C, (dot‐dashed lines) actin rehydrated after 8 weeks at 60°C. Journal of Pharmaceutical Sciences 2000 89, 199-214DOI: (10.1002/(SICI)1520-6017(200002)89:2<199::AID-JPS7>3.0.CO;2-B) Copyright © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association Terms and Conditions

Figure 4 (A) Loss of functional actin due to aggregate formation in rehydrated samples after storage in dextran alone expressed as percent of total protein remaining in supernatant after first centrifugation. (B) Polymerization activity expressed as percent of non‐aggregated protein sedimented after addition of salts to stimulate filament assembly. Filled symbols indicate storage at 45°C, open symbols indicate storage at 60°C. The interval bounded by dotted lines represents the 95% confidence interval for the polymerization of native actin (n = 3). Journal of Pharmaceutical Sciences 2000 89, 199-214DOI: (10.1002/(SICI)1520-6017(200002)89:2<199::AID-JPS7>3.0.CO;2-B) Copyright © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association Terms and Conditions

Figure 5 Area normalized, second‐derivative amide I spectra of actin dried with disaccharides: (dotted lines) native actin, (solid lines) spectra taken immediately after freeze‐drying, (dashed lines) spectra taken after 8 weeks at 45°C, (dot‐dashed lines) spectra taken after 8 weeks at 60°C. (A) 1% Sucrose, (B) 5% sucrose, (C) 1% trehalose, (D) 5% trehalose. Journal of Pharmaceutical Sciences 2000 89, 199-214DOI: (10.1002/(SICI)1520-6017(200002)89:2<199::AID-JPS7>3.0.CO;2-B) Copyright © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association Terms and Conditions

Figure 6 Circular dichroism spectra of rehydrated actin after drying with (A) 1% sucrose, (B) 5% sucrose, (C) 1% trehalose, or (D) 5% trehalose; (dotted lines) native actin, (solid lines) actin rehydrated immediately after freeze‐drying, (dashed lines) actin rehydrated after 8 weeks at 45°C, (dot‐dashed lines) actin rehydrated after 8 weeks at 60°C. Journal of Pharmaceutical Sciences 2000 89, 199-214DOI: (10.1002/(SICI)1520-6017(200002)89:2<199::AID-JPS7>3.0.CO;2-B) Copyright © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association Terms and Conditions

Figure 7 (A) Loss of functional actin due to aggregate formation in rehydrated samples after storage expressed as percent of total protein remaining in supernatant after first centrifugation. (Circles) Actin dried with sugar only, (triangles) actin dried with sugar plus dextran; filled symbols indicate storage at 45°C, open symbols indicate storage at 60°C. (A) 1% Sucrose, (B) 5% sucrose, (C) 1% trehalose, (D) 5% trehalose. Journal of Pharmaceutical Sciences 2000 89, 199-214DOI: (10.1002/(SICI)1520-6017(200002)89:2<199::AID-JPS7>3.0.CO;2-B) Copyright © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association Terms and Conditions

Figure 8 Area normalized, amide I infrared spectra of (solid line) aggregated actin formed after rehydration of protein dried with disaccharides, (dashed line) aggregated actin induced by heating protein solution to 100°C. Dotted line is native actin. Journal of Pharmaceutical Sciences 2000 89, 199-214DOI: (10.1002/(SICI)1520-6017(200002)89:2<199::AID-JPS7>3.0.CO;2-B) Copyright © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association Terms and Conditions

Figure 9 Polymerization activity expressed as percent of non‐aggregated protein sedimented after addition of salts to stimulate filament assembly. (Circles) Actin dried with sugar only, (triangles) actin dried with sugar plus dextran; filled symbols indicate storage at 45°C, open symbols indicate storage at 60°C. The interval bounded by dotted lines represents the 95% confidence interval for the polymerization of native actin (n = 3). (A) 1% Sucrose, (B) 5% sucrose, (C) 1% trehalose, (D) 5% trehalose. Journal of Pharmaceutical Sciences 2000 89, 199-214DOI: (10.1002/(SICI)1520-6017(200002)89:2<199::AID-JPS7>3.0.CO;2-B) Copyright © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association Terms and Conditions

Figure 10 Area‐normalized, second‐derivative amide I spectra of actin dried with disaccharides plus dextran: (dotted lines) native actin, (solid lines) spectra taken immediately after freeze‐drying, (dashed lines) spectra taken after 8 weeks at 45°C, (dot‐dashed lines) spectra taken after 8 weeks at 60°C. (A) 1% Sucrose, (B) 5% sucrose, (C) 1% trehalose, (D) 5% trehalose. Journal of Pharmaceutical Sciences 2000 89, 199-214DOI: (10.1002/(SICI)1520-6017(200002)89:2<199::AID-JPS7>3.0.CO;2-B) Copyright © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association Terms and Conditions

Figure 11 Circular dichroism spectra of rehydrated actin after drying with dextran plus: (A) 1% sucrose, (B) 5% sucrose, (C) 1% trehalose, or (D) 5% trehalose; (dotted lines) native actin, (solid lines) actin rehydrated immediately after freeze‐drying, (dashed lines) actin rehydrated after 8 weeks at 45°C, (dot‐dashed lines) actin rehydrated after 8 weeks at 60°C. Journal of Pharmaceutical Sciences 2000 89, 199-214DOI: (10.1002/(SICI)1520-6017(200002)89:2<199::AID-JPS7>3.0.CO;2-B) Copyright © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association Terms and Conditions