This work focuses on the effects of formulation and manufacturing as it effects protein structure and physical stability. Using common physical characterization techniques, X-ray photoelectron spectroscopy, and solid-state hydrogen/deuterium exchange with mass spectrometry, correlations are identified between these results and accelerated stability studies.

<div><div><div><p>To prolong their long-term stability, protein molecules are commonly dispensed as lyophilized powders to be reconstituted before use. Evaluating the stability of these biomolecules in the solid state is routinely done by using various analytical techniques such as glass transition temperature, residual moisture content and other spectroscopic techniques. However, these techniques often show poor correlation with long term storage stability studies. As a result, time intensive long term storage stability studies are still the golden standard for evaluating protein formulations in the solid state. Over the past few years, our lab has developed solid-state hydrogen deuterium exchange- mass spectrometry (ssHDX-MS) as an analytical tool that probes the backbone of a protein molecule in the solid state. ssHDX-MS gives a snapshot of protein-matrix interactions in the solid state and has a quick turnaround of a few weeks as opposed to a few months for accelerated stability testing. Additionally, various studies in the past have demonstrated that ssHDX-MS can be used for a wide range of biomolecules and shows strong correlation to long term stability studies routinely employed.</p><p>The main aim of this dissertation is to provide an initial understanding of the mechanism behind ssHDX-MS in structured protein formulations. Specifically, this dissertation is an attempt at studying the effects of various experimental variables on the ssHDX-MS of myoglobin formulations as well as demonstrating the utility of this analytical technique. Firstly, the effects of varying temperature and relative humidity on ssHDX-MS of myoglobin formulations is studied with the help of statistical modeling. Secondly, the effects of pressure on ssHDX-MS of myoglobin formulations are evaluated at an intact and peptide digest levels. Finally, ssHDX-MS is used as a characterization tool to evaluate the effects of two different lyophilization methods on the structure and stability of myoglobin formulations. The results of studies described in this dissertation show ssHDX-MS to be sensitive to changes in experimental parameters, namely temperature, relative humidity, pressure, and excipients. Additionally, ssHDX-MS results were in good agreement with other routinely employed analytical and stability testing techniques when used to compare the effects of two lyophilization methods on myoglobin formulations.</p></div></div></div>

<p>  </p> <p>With the recent growth in demand for biologics across the globe, it remains critical to manufacture these biologics in solid-state to improve stability as well as to increase the ease of transportation across the world. To meet these increased demands, it is of paramount importance to use various processing methods that have shorter processing times. It is also important to understand the impact of the processing methods and various formulation components on the stability of the proteins.  In Chapter 1, a review of the various processing methods that are used in the industry along with additional processing methods that are being investigated will be discussed. The common drying methods such as lyophilization and spray drying along with the novel techniques as well as specific examples of processing parameters to improve the processing conditions that better suit the protein formulations will be mentioned. </p> <p>The studies in Chapter 2 examined the effects of processing methods (freeze drying and spray freeze drying) and the excipients on the protein structure and physical stability. Protein solids containing one of two model proteins (lysozyme or myoglobin) were produced with or without excipients (sucrose or mannitol) using freeze drying or spray freeze drying (SFD). The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), circular dichroism spectrometry (CD), size exclusion chromatography (SEC), BET surface area measurements, and solid-state hydrogen-deuterium exchange with mass spectrometry (ssHDX-MS). ssFTIR and CD could identify little to no difference in the structure of the proteins in the formulation. ssHDX-MS was able to identify the population heterogeneity, which was undetectable by conventional characterization techniques of ssFTIR and CD. ssHDX-MS metrics such as Dmax and peak area showed a good correlation with the protein physical instability (loss of the monomeric peak area by size exclusion chromatography) in 90-day stability studies conducted at 40oC for lysozyme.  The higher specific surface area was associated with greater loss in monomer content for myoglobin-mannitol formulations as compared to myoglobin-only formulations. Spray freeze drying seems a viable manufacturing technique for protein solids with appropriate optimization of formulations. The differences observed within the formulations and between the processes using ssHDX-MS, BET surface area measurements, and SEC in this study provide an insight into the influence of drying methods and excipients on protein physical stability.</p> <p>Based on this work, it was identified that spray freeze drying can be a viable alternative to produce solid-state protein formulations with similar stability as the freeze drying process. However, due to the long processing times and scale-up issues involved in the spray freeze drying process, there is a necessity to explore additional drying processes. Chapter 3 focuses on using another novel technique known as electrostatic spray drying (ESD) to produce solid-state protein formulations at lower drying temperatures than conventional spray drying and its effect on protein stability. A mAb formulation was dried by either conventional spray drying or electrostatic spray drying with charge (ESD). The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), size exclusion chromatography (SEC), and solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). Particle characterizations such as BET surface area, particle size distribution, and particle morphology were also performed. Conventional spray drying of the mAb formulation at the inlet temperature of 70oC failed to generate dry powders due to poor drying efficiency; electrostatic spray drying at the same temperature at 5kV enabled the formation of powder formulation with satisfactory moisture contents. Deconvoluted peak areas of deuterated samples from the ssHDX-MS study showed a good correlation with the loss of the monomeric peak area measured by size exclusion chromatography in the 90-day accelerated stability study conducted at 40oC. Low-temperature (70oC inlet temperature) drying with an electrostatic charge (5kV) led to better protein physical stability as compared with the samples spray-dried at the high temperature (130oC inlet temperature) without charge.</p> <p>This study shows that electrostatic spray drying can produce solid monoclonal antibody formulation at a lower inlet temperature than traditional spray drying with better physical stability. While ESD can be a viable option for thermal-sensitive formulations, it is important to understand the impact of various formulation components on the stability of the proteins while using spray drying. Based on our previous studies, a good understanding of the effect of different sugars and the presence of surfactants on the spray-dried proteins has been established. However, the impact of the selection of buffer on protein stability has not been studied. In Chapter 4, the effect of buffer salts on the physical stability of spray dried and lyophilized formulations of a model protein, bovine serum albumin (BSA) were examined. BSA formulations with various buffers were dried by either lyophilization or spray drying. The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), powder X-ray diffraction (PXRD), size exclusion chromatography (SEC), solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS), and solid-state nuclear magnetic resonance spectroscopy (ssNMR). Particle characterizations such as BET surface area, particle size distribution, and particle morphology were also performed. Results from conventional techniques such as ssFTIR did not exhibit correlations with the physical stability of studied formulations. Deconvoluted peak areas of deuterated samples from the ssHDX-MS study showed a satisfactory correlation with the loss of the monomeric peak area measured by SEC (R2 of 0.8722 for spray-dried formulations and 0.8428 for lyophilized formulations) in the 90-day accelerated stability study conducted at 40oC. PXRD was unable to measure phase separation in the samples right after drying. In contrast, ssNMR successfully detected the occurrence of phase separation between the succinic buffer component and protein in the lyophilized formulation, which results in a distribution of microenvironmental acidity and the subsequent loss of long-term stability. In summary, this study demonstrated that buffer salts have less impact on physical stability for the spray-dried formulations than the lyophilized solids.</p> <p>The study in Chapter 5 looked at examining the physical stability of spray freeze dried (SFD) bovine serum albumin (BSA) solids produced using the radio frequency (RF)-assisted drying technique. BSA formulations were prepared with varying concentrations of trehalose and mannitol, with an excipient-free formulation as control. These formulations were produced using traditional spray freeze drying (SFD) or RF-assisted spray freeze drying (RFSFD). The dried formulations were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), Karl Fischer moisture content measurement, powder X-ray diffraction (PXRD), size exclusion chromatography (SEC), solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). Traditional characterization tools such as ssFTIR and moisture content did not have a good correlation with the physical stability of the formulations measured by SEC. ssHDX-MS metrics such as the maximum deuterium uptake (Dmax) (R2 = 0.791) and deconvoluted peak areas of the deuterated samples (R2 = 0.914) showed a satisfactory correlation with the SEC stability data. RFSFD improved the stability of formulations with 20 mg/ml of trehalose and no mannitol and had similar stability with all other formulations as compared to SFD. This study demonstrated that the RFSFD technique can significantly reduce the duration of primary drying cycle from 48 h to 27.5 h while maintaining or improving protein physical stability as compared to traditional lyophilization.</p> <p>Lastly, Chapter 6 consists of a summary of the conclusions formed from the work presented in this thesis. Furthermore, suggestions for future work are provided based on observations of results, less-explored areas of formulation and processing conditions as well as characterization tools to understand effects on protein physical stability.</p> <p><br></p>