Journal: Journal of pharmaceutical sciences

Article Title: Process and Formulation Effects on Protein Structure in Lyophilized Solids using Mass Spectrometric Methods

doi: 10.1016/j.xphs.2016.02.033

Figure Lengend Snippet: Product temperature profiles for excipient-free (Mb-A) and sucrose-containing (Mb-B) myoglobin lyophilized without controlled nucleation (panel A) and with controlled nucleation (panel B). Thermocouple probes (TC) were placed inside the vial (labeled Mb-A in, Mb-B in) or taped to the outside of the vial (labeled Mb-A out, Mb-B out). The shelf set point (shelf SP) temperature is represented by the black line in panels A and B.

Article Snippet: For freezing with controlled nucleation (LyoStar 3 with ControLyo ® , SP Industries, Inc., Gardiner, NY), the vials were equilibrated at 5 °C for 30 min, followed by pressurization with argon gas to 28 psig and a decrease in temperature to −5 °C.

Techniques: Labeling

Journal: Journal of pharmaceutical sciences

Article Title: Process and Formulation Effects on Protein Structure in Lyophilized Solids using Mass Spectrometric Methods

doi: 10.1016/j.xphs.2016.02.033

Figure Lengend Snippet: Extent of supercooling for excipient-free (Mb-A, panel A) and sucrose-containing (Mb-B, panel B) myoglobin formulations lyophilized without controlled nucleation. Thermocouples were placed inside the vial in contact with the solution and also taped to the outside of the same vial. Mb-A showed supercooling to ~ −7.5 ± 0.4 °C and Mb-B showed supercooling to ~ −9.1 ± 0.4 °C as measured by thermocouples placed both inside and outside the vial.

Article Snippet: For freezing with controlled nucleation (LyoStar 3 with ControLyo ® , SP Industries, Inc., Gardiner, NY), the vials were equilibrated at 5 °C for 30 min, followed by pressurization with argon gas to 28 psig and a decrease in temperature to −5 °C.

Techniques:

Journal: Journal of pharmaceutical sciences

Article Title: Process and Formulation Effects on Protein Structure in Lyophilized Solids using Mass Spectrometric Methods

doi: 10.1016/j.xphs.2016.02.033

Figure Lengend Snippet: Second-derivative solid-state FTIR spectra for excipient-free (Mb-A) and sucrose-containing (Mb-B) lyophilized myoglobin formulations. Spectra were obtained for Mb-A and Mb-B formulations lyophilized with controlled (black and gray continuous curves respectively) and without controlled nucleation (black and gray dashed curves respectively).

Article Snippet: For freezing with controlled nucleation (LyoStar 3 with ControLyo ® , SP Industries, Inc., Gardiner, NY), the vials were equilibrated at 5 °C for 30 min, followed by pressurization with argon gas to 28 psig and a decrease in temperature to −5 °C.

Techniques:

Journal: Journal of pharmaceutical sciences

Article Title: Process and Formulation Effects on Protein Structure in Lyophilized Solids using Mass Spectrometric Methods

doi: 10.1016/j.xphs.2016.02.033

Figure Lengend Snippet: SEM images of excipient-free (Mb-A; panels A, B) and sucrose-containing (Mb-B; panels C, D) lyophilized myoglobin formulations. Panels A, C: Formulations lyophilized with controlled nucleation; Panels B, D: Formulations lyophilized without controlled nucleation. The top and bottom of each image represent the top and bottom of the cake respectively. Scale bars are set at 1 mm.

Article Snippet: For freezing with controlled nucleation (LyoStar 3 with ControLyo ® , SP Industries, Inc., Gardiner, NY), the vials were equilibrated at 5 °C for 30 min, followed by pressurization with argon gas to 28 psig and a decrease in temperature to −5 °C.

Techniques:

Journal: Journal of pharmaceutical sciences

Article Title: Process and Formulation Effects on Protein Structure in Lyophilized Solids using Mass Spectrometric Methods

doi: 10.1016/j.xphs.2016.02.033

Figure Lengend Snippet: Deconvoluted mass spectra for excipient-free (Mb-A) and sucrose-containing (Mb-B) myoglobin formulations after 48 h of solid-state hydrogen-deuterium exchange at 5 °C, 43 % RH. Mass spectra were obtained for Mb-A and Mb-B lyophilized with controlled nucleation (black and gray continuous curves respectively) and without controlled nucleation (black and gray dashed curves respectively). The dashed and dotted curve represents the deconvoluted spectrum for undeuterated protein.

Article Snippet: For freezing with controlled nucleation (LyoStar 3 with ControLyo ® , SP Industries, Inc., Gardiner, NY), the vials were equilibrated at 5 °C for 30 min, followed by pressurization with argon gas to 28 psig and a decrease in temperature to −5 °C.

Techniques:

Journal: Journal of pharmaceutical sciences

Article Title: Process and Formulation Effects on Protein Structure in Lyophilized Solids using Mass Spectrometric Methods

doi: 10.1016/j.xphs.2016.02.033

Figure Lengend Snippet: Deuterium uptake kinetics for excipient-free myoglobin (Mb-A) lyophilized with and without controlled nucleation (closed and open circles respectively) and myoglobin-sucrose formulation (Mb-B) lyophilized with and without controlled nucleation (closed and open triangles respectively). Each point represents the average number of deuterons (n = 3, ± SD) incorporated after incubation at 43 % RH, 5 °C for 0.5, 2, 6, 12, 24, 48, 84 and 120 h.

Article Snippet: For freezing with controlled nucleation (LyoStar 3 with ControLyo ® , SP Industries, Inc., Gardiner, NY), the vials were equilibrated at 5 °C for 30 min, followed by pressurization with argon gas to 28 psig and a decrease in temperature to −5 °C.

Techniques: Formulation, Incubation

Journal: Journal of pharmaceutical sciences

Article Title: Process and Formulation Effects on Protein Structure in Lyophilized Solids using Mass Spectrometric Methods

doi: 10.1016/j.xphs.2016.02.033

Figure Lengend Snippet: ssHDX-MS peak widths for myoglobin formulations in the absence (Mb-A) and presence (Mb-B) of sucrose lyophilized with or without controlled nucleation. Peak widths at ~28 % deuteration were calculated from the deconvoluted mass spectra for each formulation at 20 % peak height.

Article Snippet: For freezing with controlled nucleation (LyoStar 3 with ControLyo ® , SP Industries, Inc., Gardiner, NY), the vials were equilibrated at 5 °C for 30 min, followed by pressurization with argon gas to 28 psig and a decrease in temperature to −5 °C.

Techniques: Formulation

Journal: Journal of pharmaceutical sciences

Article Title: Process and Formulation Effects on Protein Structure in Lyophilized Solids using Mass Spectrometric Methods

doi: 10.1016/j.xphs.2016.02.033

Figure Lengend Snippet: (A) Deconvoluted mass spectra for unlabeled myoglobin (dotted line) and myoglobin labeled with photo-leucine (solid line). The spectrum for labeled myoglobin has been offset vertically to show differences. Peaks corresponding to the mass of unlabeled (0 L, ~ 16951 Da) and protein with 1 label (1 L, ~ 17066 Da) were detected. A peak for myoglobin labeled with 2 labels (~ 17182 Da) was also detected, but not shown here since the intensity was low. Similar spectra with up to 2 labels were obtained for excipient-free (Mb-A) and sucrose-containing (Mb-B) myoglobin formulations lyophilized with and without controlled nucleation. (B) Fraction of protein labeled with photo-leucine for Mb-A and Mb-B formulations lyophilized with controlled nucleation (CN) or without controlled nucleation (UCN). The fraction was calculated using peak heights of labeled protein on the extracted ion chromatogram (EIC) obtained by LC-MS. The bars show the mean (± SD) of three LC-MS injections. The following pairs showed significantly different means (p < 0.05, GraphPad Prism) using one-way ANOVA: Mb-A CN vs. Mb-B CN and Mb-B CN vs. Mb-B UCN.

Article Snippet: For freezing with controlled nucleation (LyoStar 3 with ControLyo ® , SP Industries, Inc., Gardiner, NY), the vials were equilibrated at 5 °C for 30 min, followed by pressurization with argon gas to 28 psig and a decrease in temperature to −5 °C.

Techniques: Labeling, Liquid Chromatography with Mass Spectroscopy

Journal: Journal of pharmaceutical sciences

Article Title: Process and Formulation Effects on Protein Structure in Lyophilized Solids using Mass Spectrometric Methods

doi: 10.1016/j.xphs.2016.02.033

Figure Lengend Snippet: Peptide-level labeling with photo-leucine for (A) excipient-free (Mb-A) myoglobin lyophilized with controlled nucleation. (B) Sucrose-containing (Mb-B) myoglobin lyophilized with controlled nucleation Labeled peptides Leu32-Lys42 and Tyr103-Lys118 are represented in blue and the heme group is shown in red. Mb-A and Mb-B lyophilized without controlled nucleation showed the same labeling pattern on Tyr103-Lys118 as represented in panel (A). The crystal structure for holomyoglobin was obtained from the Protein Data Bank (www.rcsb.org; PDB ID 1WLA) and PyMOL was used to generate labeled ribbon diagrams (PyMOL Molecular Graphics System, Version 1.4.1, Schrödinger, LLC).

Article Snippet: For freezing with controlled nucleation (LyoStar 3 with ControLyo ® , SP Industries, Inc., Gardiner, NY), the vials were equilibrated at 5 °C for 30 min, followed by pressurization with argon gas to 28 psig and a decrease in temperature to −5 °C.

Techniques: Labeling