Supplementary Material

Salt formation during freeze-drying of tris buffered solution- an approach to enhance indomethacin dissolution

Seema Thakral and Raj SuryanarayananDepartment of Pharmaceutics, University of Minnesota Minneapolis MN 55455 USA

Section S1. Thermal analysis of prelyo solution

Fig. S1. DSC heating curves of frozen prelyophilization solution (0.1 M). Effect of heating rate.Fig S2. DSC heating curves of frozen prelyophilization solutions. Effect of solute concentration. Fig. S3. DSC heating curve of frozen tris (0.05 M) solution. Fig. S4. Effect of annealing on the glass transition temperature of frozen prelyophilization solution.

Section S2. Preparation and characterization of ‘neat’ IMC-tris saltFig. S5. IR spectra of the ‘neat’ IMC-tris salt and IMC-tris lyophile.Fig. S6. Overlay of XRD pattern of IMC-tris salt and lyophile obtained after annealing. Fig. S7. DSC heating curves of IMC-tris salt and of lyophile obtained after annealing.

Section S3. Thermal analysis of lyophile (obtained after annealing the frozen prelyo

solution)

Fig. S8. X-ray diffraction patterns of the lyophile heated from RT, first to 60 ºC and then progressively to 80, 100 and 120 ºC. Fig. S9. Isothermal thermogravimetric analysis of lyophile. Fig. S10. IR spectra of lyophile subjected a controlled temperature programs in the TGA.

Section S1: Thermal analysis of prelyo solution

Fig S1. DSC heating curves of frozen prelyophilization solution (0.1 M). Each solution was cooled from RT to -50 °C at 1 °C/min, held for 30 min and heated to 25 °C at:(A) 0.5 ºC/min, (B) 1 ºC/min and (C) 10 ºC/min. Only the heating curve is shown. There was no evidence of solute crystallization.

Fig. S2. DSC heating curve of frozen prelyo solutions*. Each solution was cooled from RT to -50 °C at 1 °C/min, held for 30 min and heated to 25 °C at 10 °C/min. The glass transition temperature of the freeze concentrate (Tgʹ) is shown in the expanded inset. Only the heating curve is shown. *The concentration, specified next to each DSC curve, is with respect to IMC.

Fig. S3. DSC heating curve of frozen tris (0.05 M) solution. The solution was cooled from RT to -90 °C at 1 °C/min, held for 30 min, and heated to 25 °C at 10 °C/min. The endotherm attributable to eutectic melting is evident at -5 ºC. The inset shows an exotherm ascribed to crystallization at ~ -25 ºC.

Fig. S4. Tgʹ (n=3) of frozen prelyophilization solution (0.1 M) as a function of annealing temperature and time. The experimental details are provided in the Methods Section.

-25 -20 -15 -10 -5 0-35

-34

-33

-32

-31

0 h

1h

3 h

5 h

Annealing temperature, 0C

Tg'

, ºC

Section S2. Preparation and characterization of IMC-tris saltIMC (358 mg) and tris (121 mg) was separately dissolved in acetone (10 mL and 100 mL respectively). The solutions were mixed and sonicated for 10 min. The solid residue left after solvent evaporation, hereafter referred to as IMC-tris salt, was characterized by IR spectroscopy, X-ray diffractometry and differential scanning calorimetry.

Fig. S5. IR spectra of IMC-tris salt and IMC-tris lyophile (1900 – 1370 cm-1 region). Vertical broken lines: (a) asymmetric stretch due to carboxylate anion, and (b) symmetric stretch due to carboxylate anion (observed as a weak shoulder).

Fig. S6. XRD patterns of (A) lyophile obtained after annealing and (B) IMC-tris salt.

Fig. S7. DSC heating curves of (A) lyophile obtained after annealing and (B) IMC-tris salt.

Summary of the results. IR spectroscopy confirmed salt formation. (IMC was present in the “ionized state” in the salt). The XRD pattern of the lyophile revealed lines attributable to IMC-tris salt, with d-spacings (Å) of: 10.1 (8.74 °2θ), 6.79 (13.1 °2θ), 3.48 (25.5 °2θ) and , 3.29 (26.5 °2θ). The DSC heating curve of IMC-tris salt shows an endotherm at 146 ºC, attributed to melting of the salt. The melting endotherm in the lyophile was observed at a lower temperature (140 ºC), attributable to the presence of ‘excess’ tris in the lyophile.

Section S3. Thermal analysis of the lyophile (obtained after annealing the frozen prelyo

solution)

Introduction. The lyophile obtained after annealing the frozen solution revealed several thermal events - an endotherm at ~ 87 ºC, an exotherm at ~ 100 ºC followed by an endotherm at ~ 141 ⁰C. The endotherm at ~ 87 ºC could be attributed to water loss. In order to characterize the other thermal events, variable temperature XRD, thermogravimetric analysis and IR studies were conducted.

MethodsVariable temperature X-ray diffractometry. About 50 mg of lyophile was packed (top filling) into a copper holder and the stage was heated from RT to the desired temperature at 10 ºC/min. During the XRD scan, the sample at the selected temperature was maintained under isothermal conditions. The other experimental details are provided in the Methods section.

Thermogravimetric analysis. In a thermogravimetric analyzer (Model Q500, TA instruments), 4–6 mg of sample was heated in an open pan from RT to the desired temperature, at 10 ºC/min, under dry nitrogen purge and held isothermally for the desired time period. It was then subjected to IR spectroscopy.

Fig. S8: X-ray diffraction patterns of the lyophile heated from RT, first to 60 ºC (a) and then progressively to 80 (b), 100 (c) and 120 ºC (d). The sample was held for 15 min at each temperature. It was finally heated to 140 ºC (e). The heating rate was 10 ºC/min and the sample was held under isothermal conditions during the XRD run.

Fig. S9. Isothermal thermogravimetric analysis of lyophile. (a) 60 ºC for 30 min; weight loss 0.50%; (b) 80ºC for 15 min; weight loss 1.2%; (c) 100 ºC for 10 min; weight loss 1.2%; (d) 120 ºC for 5 min; weight loss 1.5%; (e) 140 ºC for 5 min; weight loss 3.3%.

Fig. S10: IR spectra of lyophile subjected a controlled temperature programs in the TGA. The TGA experimental conditions are given in Fig. S5.

Summary of the results. In the IR spectra of lyophiles (Fig. S10) heated to different temperatures, the emergence (at 100 ºC) and subsequent increase (at 120 ºC) in peak intensities at 1640 cm-1 and at 1540 cm-1

suggest a chemical change. In the variable temperature XRD, new peaks appeared at 100 ºC and their intensities increased at 120 ºC (Fig. S8). Thus, the high temperature events in the heating curve (Fig. 6 in the main manuscript; thermal events observed at ≥ 100 ºC) may be attributed to a combination of physical and chemical transformations. Further characterization was beyond the scope of this study.