200 research outputs found
Modelling climate change impacts on European grassland-based livestock systems
No full textClimate change is leading to higher temperatures and altered rainfall patterns across Europe. These changes are likely to have major impacts on plant life. This is particularly relevant for livestock production systems which are dependent on grass and forage. Farmers need to know what they can expect in the future so that they can be well prepared and ensure that their livestock will have enough to eat. This thesis aims to quantify the impacts of rising atmospheric CO2 concentrations, higher temperatures and changes in water availability on the yield and protein content of European grasslands.
The first approach used was a meta-analysis. Data from experiments in which the climate had been artificially altered was collected and divided according to geographic region (Alpine, Atlantic, continental, northern and southern) and plant type (graminoids, legumes, forbs and shrubs). Using Markov Chain Monte Carlo (MCMC) simulations, mixed models were developed to estimate the expected changes to plant yield and protein (i.e. nitrogen (N)) concentration under different climatic changes. The results showed that areas predicted to become warmer and wetter (i.e. northern Europe and parts of Alpine and continental Europe) will benefit from higher plant yields, but reduced plant N concentration. Areas which will become warmer and drier (i.e. southern Europe and parts of continental Europe) will see decreases in both yield and N concentration. The Atlantic region is the area where climate change is expected to be the least extreme and the effects on plant life will be relatively minor. Shrubs will particularly benefit from rising atmospheric CO2 concentrations, though will also suffer large decreases in N concentration, as will forbs.
The next approach considered different methodologies for modelling grassland yield and N yield. One method involved developing a statistical model using data from long-term grassland experiments across Europe. Through stepwise linear regression, equations were developed to model grassland yield and N yield based on various weather and managerial variables. The other method used a pre-existing process-based model (Century), which was applied to six sites across Europe. Both approaches produced reasonable estimates of grassland yield and N yield. The prediction error was lower for the Century model while the regression methodology produced better correlations between observations and predictions. Both models were quite sensitive to uncertainties in weather parameters, particularly precipitation, with little sensitivity to soil properties. Overall, the regression approach was found to be suitable for considering general trends over large spatial scales, while the Century model was more appropriate for local-scale analysis.
The two models described above were used to quantify the effects of two different climate change scenarios (one midrange and one more extreme) on the five European regions listed above. The two models generally produced similar predictions, indicating that grassland yields will increase in most areas though there may be slight decreases in southern Europe. Also, plant N concentrations will decrease. Generally permanent grasslands responded more positively to climate change than temporary ones. The impact of climate change tends to be less than the impact of fertiliser, geographic region or grassland type, suggesting that appropriate changes to grassland management practices should be able to mitigate the negative effects of climate change.
The modelling described above was all performed using a monthly time-step. This is computationally efficient, but means that short-term extreme weather events are not accounted for. Extreme weather events such as heavy rainfall, droughts and heat waves are predicted to become both more frequent and more intense in the future and it is important to consider the impacts they will have on grasslands and therefore livestock.
Two methodologies were used to quantify the effects of extreme weather events on grasslands. The first uses multiple regression analysis and incorporates terms such as ‘number of days in a month with temperature greater than 30°’ to account for weather extremes. The equations developed had a good fit with observed data. They were found to be predominantly sensitive to uncertainties in precipitation rather than in temperature or grassland species composition. Two projected future weather datasets were applied to the equations; both followed the same climate change scenario, but one included extreme events and the other was smoothed to reduce the extremes. Comparing the model outputs from the two datasets showed that smoothing the data increased the predicted yields and N yields, demonstrating that extreme weather events are detrimental to grasslands. In general, the yield of temporary grasslands decreased over time, while for permanent grasslands it increased. There was little change in N yield over time.
The other methodology used the pre-existing process-based model DailyDayCent, which is very similar to the Century model, but is based on a daily rather than a monthly time-step. DailyDayCent was applied to six sites across Europe and was found to have reasonably good fit, though struggled to capture inter-annual variability. The model was predominantly sensitive to uncertainties in rainfall measurements rather than temperature. Two climate change datasets, with and without extreme events, were applied to the model for each of the six sites. Predicted yields and N yields were similar to those found with the Century model. The presence or absence of extreme events usually had little effect, but this may have been due to limitations of the model. The exception was for a site in southern Europe, where the presence of extreme events led to increases in yield and N yield in the short-term, but large decreases in the long-term.
Overall, grassland yields are expected to increase in the future in response to climate change (except possibly in southern Europe), particularly for permanent grasslands, while plant N concentration will decrease. Increased yields are generally good for livestock, though reduced N concentrations indicate that grazing animals will need to have a higher intake in order to receive the same amount of protein. Extreme weather events are an important consideration, leading to reductions in grassland yield and N yield. Farmers need to be prepared to meet the challenges presented by such events, for example through using more resilient plant species or increasing plant species richness
Long-term antibiotic exposure in soil is associated with changes in microbial community structure and prevalence of class 1 integrons
No full textAntimicrobial resistance is one of the most significant challenges facing the global medical community and can be attributed to the use and misuse of antibiotics. This includes use as growth promoters or for prophylaxis and treatment of bacterial infection in intensively farmed livestock from where antibiotics can enter the environment as residues in manure. We characterised the impact of the long-term application of a mixture of veterinary antibiotics alone (tylosin, sulfamethazine and chlortetracycline) on class 1 integron prevalence and soil microbiota composition. Class 1 integron prevalence increased significantly (P < 0.005) from 0.006% in control samples to 0.064% in the treated plots. Soil microbiota was analysed using 16S rRNA gene sequencing and revealed significant alterations in composition. Of the 19 significantly different (P < 0.05) OTUs identified, 16 were of the Class Proteobacteria and these decreased in abundance relative to the control plots. Only one OTU, of the Class Cyanobacteria, was shown to increase in abundance significantly; a curiosity given the established sensitivity of this class to antibiotics. We hypothesise that the overrepresentation of Proteobacteria as OTUs that decreased significantly in relative abundance, coupled with the observations of an increase in integron prevalence, may represent a strong selective pressure on these taxa
Computational modelling of protein fibrillation with application to glucagon
No full textA computational method to model the steric zipper of amyloid fibrils (FibPreditor) is developed. The method generates an ensemble of structures for the steric zipper by a number of geometric operations and presents the most energetically favorable candidates as models of steric zipper. The method is shown to successfully reproduce a number of experimentally determined fibril structures. FibPredictor is then applied to model the steric zipper of glucagon fibrils. Phosphate ester derivatives of glucagon are designed based on these models as soluble and stable prodrugs or active alternatives for glucagon. A number of penta-peptide chaperones are also designed as excipients to delay glucagon fibrillation. Although penta-peptides can delay glucagon fibrillation, they are less effective compared to phosphorylation of glucagon
Photolytic Labeling to Probe Peptide-Matrix Interactions in Lyophilized Solids
No full textTherapeutic proteins are often lyophilized with excipients such as sucrose or trehalose to protect them during manufacturing and achieve a longer shelf-life. Formulation design for therapeutic proteins has been a trial-and-error process, and the mechanisms responsible for the stabilizing effects of excipients are not fully understood. Two proposed theories have been widely accepted: the water replacement theory and the vitrification theory.1,2 The water replacement theory suggests that excipients stabilize protein molecules in the solid state by forming hydrogen bonds that “replace” the hydrogen bonds to water that stabilize the protein in solution, while the vitrification theory asserts that proteins are stabilized by a glassy solid matrix of low mobility and does not require direct interactions between excipient and protein. A better understanding of the interactions between proteins and other components of the lyophilized matrix can facilitate rational formulation design and shorten the time in development. However, most of the analytical methods available can only provide information on the bulk properties of the lyophilized matrix such as moisture content and glass transition temperature (Tg); it has been difficult to measure the interactions between protein and excipient directly, if they exist. In order to characterize the interactions between protein and excipients in a lyophilized matrix with high resolution, a photolytic labeling method was developed in this dissertation, building on previous work in our research group. Photolytic labeling has long been used to identify protein-protein interactions in vivo. 3,4 Common types of photo-reaction reagents and their applications are summarized in Chapter 1. The research described in this dissertation utilizes the diazirine functional group, which is activated after UV exposure and undergoes a free radical reaction to form covalent bonds with nearby molecules. The reaction can be used to identify the interactions between excipients and protein or peptide in a solid formulation. Previous studies in our lab have shown that photo-reaction can be applied to lyophilized solids to study protein-matrix properties and interactions in the solid. 5,6 This dissertation seeks to further identify photo-reaction products and analyze them in a more quantitative way.Chapter 2 describes a quantitative analysis of photo-reaction products in solution and lyophilized solids using a model peptide, KLQ (Ac-QELHKLQ-NHCH3). The purpose of the work in this chapter is to establish a quantitative analytical method for photo-reaction products, enabling studies of peptide-excipient interactions in lyophilized solids. KLQ was derivatized with a bifunctional probe NHS-diazirine (succinimidyl 4,4’-azipentanoate; SDA) at Lys5 to be photoreactive. The SDA derivatized KLQ (KLQ-SDA) was used to study the photo-reaction products and examine excipient interactions. Identification and quantitation of photo-reaction products of KLQ-SDA was achieved with liquid chromatography mass spectrometry (LC-MS) and reversed phase HPLC (rp-HPLC). Important reaction products such as peptide-excipient adducts and peptide water adducts varied in different formulations. Unexpected reaction products such as unproductive “dead-end” products and peptide-phosphate adducts from buffer salt were also detected and quantified. Together, the photo-reaction products reflected the local environment near Lys5 of the peptide in the solid state. This study has provided a better understanding of photoreaction with diazirine in the lyophilized solids together with a quantitative description of the local environment near Lys5
Effects of Formulation and Manufacturing Conditions on Protein Structure and Physical Stability
No full textWith expanding interest in the use of novel processing methods for biologics, it remains critical to develop formulations capable of stabilizing the conformational state of proteins and ensuring long-term physical stability. Under manufacturing conditions, proteins are exposed to a variety of stresses that can be detrimental to the physical stability of their native structure. In Chapter 1, a review of the effects of physical stresses induced by manufacturing methods will be discussed, with emphasis on their effect on initiating denaturation and aggregation. The common physical stresses discussed will include temperature, surface-induced stresses, pH effects, freezing, dehydration, and pressure. Specific examples of degradation under these stresses will be mentioned, with formulation approaches that can be used to protect against these factors.Studies in Chapter 2 examined the effects of formulation and manufacturing methods (lyophilization and spray drying) on protein structure and physical stability. Powders containing one of four model proteins (myoglobin, bovine serum albumin, lysozyme, β-lactoglobulin) were formulated with either sucrose, trehalose, or mannitol and dried using lyophilization or spray drying. The powders were characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), solid-state fluorescence spectroscopy, differential scanning calorimetry (DSC) and solid-state hydrogen/ deuterium exchange mass spectrometry (ssHDX-MS). SsFTIR and fluorescence spectroscopy identified minor structural differences among powders with different excipients and drying methods for some proteins. Using ssHDX-MS, differences were observed among protein formulations containing sucrose or trehalose and mannitol, and/or with varying processing conditions, including proteins like β-lactoglobulin, for which standard characterization techniques showed no differences. Proteins processed by spray drying typically showed greater heterogeneity by ssHDX-MS than those lyophilized; these differences were not detected by ssFTIR or solid-state fluorescence spectroscopy. The ssHDX-MS metrics were better correlated with protein physical instability measured by size-exclusion chromatography in 90- day stability studies (40°C, 33% RH) than with the results of DSC, ssFTIR, or fluorescence spectroscopy. Thus, ssHDX-MS detected subtle changes in conformation and/or matrix interactions for these proteins that were correlated with storage stability, suggesting that the method can be used to design robust solid-state protein drug products and processing methods more rapidly.From this work, it was established that population heterogeneity in spray-dried formulations was higher than those that were lyophilized, potentially due to the impact of the air-liquid interface. In order to investigate how excipients can influence the composition of protein at the surface and population heterogeneity, Chapter 3 and 4 focus on spray drying of proteins formulated with sugar-containing excipients and on the effects of surfactant inclusion, respectively. For examining the impact of saccharide-containing formulations, spray-dried formulations of myoglobin or BSA were prepared without excipient or with sucrose, trehlaose, or dextrans. Samples were characterized by ssFTIR, DSC, size exclusion chromatography (SEC), and scanning electron microscopy (SEM). Protein surface coverage was determined by X-ray photoelectron spectroscopy (XPS), while population differences were determined by ssHDX-MS. From these techniques, structural differences were exhibited with the inclusion of different excipients, with dextran formulations indicating perturbation of secondary structure. XPS indicated sucrose and trehalose reduced protein surface concentration better than dextrancontaining formulations
High-resolution mass spectrometric approaches to study protein structure and environment in lyophilized solids
No full textProteins comprise a growing class of therapeutics that is used to treat various diseases such as diabetes and cancer. However, intrinsic structural features such as the primary sequence and extrinsic factors such as pH, temperature, agitation and metal ions can promote instability that manifests as chemical degradation (e.g. oxidation, deamidation, hydrolysis) and/or physical degradation (aggregation, phase separation). Since several degradation pathways are accelerated by diffusion in solution, proteins are lyophilized to improve stability. The lyophilized formulation may still undergo degradation during manufacture and/or storage. The mechanism of protein aggregation in lyophilized solids is not well understood or predictable by conventional analytical methods such as solid-state Fourier-transform infrared spectroscopy (ssFTIR) and differential scanning calorimetry (DSC) and this poses challenges in rational formulation design. ^ This dissertation is aimed at understanding local protein structure and environment in the solid state using high-resolution mass spectrometric methods. Chapter 2 examines protein side-chain matrix accessibility using solid-state photolytic labeling- mass spectrometry (ssPL-MS). The use of a photoactive probe, photo-leucine (pLeu) enabled side-chain labeling in lyophilized formulations, reported by our group for the first time. High-resolution information at the peptide level was obtained using bottom-up tandem mass spectrometry. Differences in labeling patterns and side-chain matrix accessibility were observed when sucrose or guanidine hydrochloride was used as an excipient. This work also used a photoactive probe incorporated within the amino acid sequence of a glucagon-derived peptide to detect interactions with excipients and peptides in the solid state. Residue-level information about the preferred site of peptide-peptide crosslinking was obtained using tandem mass spectrometry. ^ Although peptide-matrix interactions could be visualized using a photoactive amino acid (PAA) derivative within the primary sequence, incorporating an unnatural amino acid into larger proteins is fairly difficult and may alter higher order structure by disturbing intra-protein contacts. Therefore, a novel photo-crosslinking method was developed to further examine the solid-state environment of lyophilized proteins, described in Chapter 3. A heterobifunctional crosslinking reagent was used to crosslink the protein with the matrix in the solid state. Some loop regions showed increased peptide-peptide adducts, while helix E showed more hydration compared to other regions. In the presence of raffinose, water replacement was not detected in the solid state; instead there was some evidence of micro-phase separation without crystallization in the solid state. Thus local protein environment in the solid state could be probed without the need for PAA incorporation within the protein sequence. ^ Lyophilization is an effective, yet expensive stabilization strategy, since conservative freeze-drying cycles often require long hours of drying. The stochastic nature of ice nucleation and lack of control over freezing can result in vial-to-vial heterogeneity due to differences in the degree of supercooling and ice crystal size. The research described in Chapter 4 focuses on using a variety of analytical methods to characterize lyophilized protein formulations to determine the effect of excipient and freezing step on protein structure. Myoglobin in the presence or absence of sucrose was lyophilized with or without controlled ice nucleation in a pilot-scale LyoStar freeze dryer. Ice nucleation occurred over a range of temperatures and times with uncontrolled nucleation, while controlled ice nucleation with rapid depressurization resulted in near-simultaneous ice nucleation. The sucrose-containing formulation showed greater retention of protein structure by ssFTIR and solid-state hydrogen-deuterium exchange mass spectrometry (ssHDX-MS). Greater conformational homogeneity was observed in the sucrose-containing formulation by ssHDX-MS peak width analysis. No significant differences in secondary structure were detected between controlled and uncontrolled nucleation using ssFTIR and ssHDX-MS. Myoglobin lyophilized with controlled nucleation in the presence of sucrose showed the greatest side-chain labeling, as determined by ssPL-MS. The results show that high-resolution mass spectrometric methods can be used to study process- and excipient effects on protein structure. ^ This thesis addresses limitations in current analytical methods used to characterize protein structure in the solid state. Whereas ssFTIR and DSC have lower sensitivity and provide information averaged over the entire sample, mass spectrometric methods can provide peptide-level information about conformational changes occurring in a small subpopulation of protein. High-resolution mass spectrometric methods have the potential to provide reliable and predictable protein formulation screening and facilitate rational drug design.
Pyroglutamate Formation and Hydrogen Deuterium Exchange in Lyophilized Therapeutic Proteins
No full textTherapeutic proteins are vital to global health, yet they are challenging to develop due to their large size and complexity. Much about their behavior is still to be understood. The research presented in this dissertation explores chemical reactions in peptides and proteins in the solid-state in order to understand formulation and matrix properties that affect reactions in solid-state therapeutic proteins. More specifically, the work applies two reactions to study reactivity: pyroglutamate (pGlu) formation and solid-state hydrogen-deuterium exchange (ssHDX). pGlu is a chemical degradant found in peptides and proteins with either glutamate (Glu) or glutamine (Gln) at the N-terminal that can occur non-enzymatically during storage. N-terminal Glu and Gln are prevalent in monoclonal antibodies (mAbs), a growing class of biologics, thus understanding this chemical modification is relevant to the development of therapeutic proteins. ssHDX with mass spectrometric analysis (ssHDX-MS) is used as an analytic tool to provide high resolution information on protein structure, stability, and matrix interactions in solid-state peptides and proteins. In this reaction deuterium donors compete for hydrogen bonding sites, which allows interrogation of the protein hydrogen bond network. While well studied when applied to the solution state, the mechanism of exchange in the solid state is not fully understood. The research and findings can be divided into three sections described below.The first section explores the mechanism of pGlu formation in the solution and solid states using a model peptide. pGlu formation and parent peptide loss were monitored by high performance liquid chromatography under accelerated storage conditions in lyophilize solid and solution formulations with vary ‘pH’ levels. ‘pH’ dependence in the solid state differed markedly from that in solution. Moreover, at the ‘pH’ where mAbs are often formulated the rate of pGlu formation is the solid state was greater than in solution.The second section aims to develop formulations that inhibit pGlu formation and to identify formulation characteristic and matrix properties that are indicative of pGlu formation. Again, a model peptide was used to monitor pGlu formation in lyophilized solid and solution formulations with varied ‘pH’ and excipients stored under accelerated conditions. The results indicate that pGlu can be inhibited by low molecular weight hydrogen bonding excipients and low moisture content.The final section aims to identify solid-state properties that affect ssHDX-MS. The effects of temperature, relative humidity (RH), and mobility on ssHDX were probed using formulations of lyophilized mAb plasticized with varying levels of glycerol. The results indicate that ssHDX kinetic parameters were influences by RH and temperature, but not glycerol level. There was a clear linear correlation with molecular mobility (T-Tg (glass transition temperature)). A first-order kinetic model was proposed that suggests a linear dependence of deuterium incorporation kinetic parameters on the product of RH and temperature, which provides a better correlation than T-Tg.The outcomes of this dissertation provide insight into solid-state reaction behavior in peptides and proteins. They have implications for the rational design of stable formulations of therapeutic proteins by expanding our understanding of a relevant chemical instability, pGlu formation, and of a high-resolution analytical technique, ssHDX-MS, that can be used to probe solid-state proteins
Thiol-disulfide exchange in human growth hormone
No full textThe biopharmaceutical industry has been growing at a tremendous rate, with sales of $63.6 billion 2012 in the US. Nevertheless, the successful development of many protein drugs has been impeded by physical and chemical instabilities arising from their inherent chemical complexity and often leading to protein aggregation. The formation of non-native disulfide bonds is a common route to covalent aggregation of therapeutic proteins and other biologics. Disulfide bonds participate in hydrolytic and oxidative degradation reactions that form non-native disulfide bonds and other reactive species. The mechanisms responsible for protein aggregation are poorly understood and formulations are currently optimized on a trial and error basis. This approach contributes to high development costs and increases the time to market. The main goal of our research is to elucidate the mechanisms of thiol-disulfide exchange and disulfide scrambling in therapeutic proteins. To accomplish this goal, model peptides derived from human growth hormone (hGH) and intact hGH were used to investigate reaction mechanisms and kinetics in solution and solid-state environments. The results will be useful in the rational development of stable, safe and efficacious protein formulations that contain free cysteines and disulfides. Chapter 1 of this dissertation focuses on background information and explains the role of disulfide bonds in proteins, their advantages and limitations and different degradation pathways. Research objective and specific aims are also outlined in Chapter 1. Model hGH-derived tryptic peptides were used to investigate reaction mechanism and kinetics in aqueous solution (Chapter 2). RP-HPLC was used as a quantitative tool and product identity was further confirmed on the LC-MS. The effects of pH, temperature, oxidation suppressants and peptide secondary structure on thiol-disulfide exchange were also explored. Protein drugs are also manufactured as lyophilized powders to improve stability and retain potency during storage. In Chapter 3 of this dissertation, thiol-disulfide exchange during lyophilization and storage in the solid state using model peptides are discussed. Comparisons are drawn to the aqueous solution studies in Chapter 2. We also investigated the effect of factors that may contribute to thiol-disulfide exchange during lyophilization and these include; initial peptide concentration, temperature, buffer type and concentration, length of primary drying time and peptide adsorption to ice. In Chapter 4, thiol-disulfide exchange in intact hGH was investigated to understand the effects of higher-order structure on reaction kinetics. Free thiol containing peptides of different length and sequence and GSH were used to facilitate thiol-disulfide exchange in intact hGH and hGH-derived peptides with a disulfide bond. Finally, concluding remarks, future perspectives and implications for protein formulations are discussed in Chapter 5
MECHANISMS AND APPLICATIONS OF SOLID-STATE HYDROGEN DEUTERIUM EXCHANGE
No full textTo 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.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.</div
Solid-State Stability of Antibody-Drug Conjugates
No full textAntibody-drug conjugates (ADCs) combine the cytotoxicity of traditional chemotherapy with the site-specificity of antibodies by conjugating payloads to antibodies with immunoaffinity. However, the conjugation alters the physicochemical properties of antibodies, increasing the risks of various types of degradation. The effects of common risk factors such as pH, temperature, and light on the stability of ADCs differ from their effects on monoclonal antibodies (mAb) due to these altered physicochemical properties.To date, ADC researchers have developed linkers with improved in vivo stability, and begun to understand the deconjugation mechanisms in vivo. In contrast, the in vitro stability of ADCs has not gained comparable attention. All nine of the U.S. FDA approved ADCs are lyophilized to minimize the potential for degradation. However, there are few studies on the solid-state stability of ADCs. To evaluate lyophilized solids, pharmaceutical development relies heavily on accelerated stability studies, which take months to determine the best formulation. Characterization methods that are often used orthogonally with accelerated studies include Fourier-transform infrared spectroscopy (FT-IR), Raman spectroscopy, near-infrared spectroscopy (NIR), differential scanning calorimetry (DSC), and x-ray powder diffraction (XRPD). Results from these methods are often poorly correlated with stability, however. Thus, stability evaluation of solid-state ADC products, and other recombinant protein drugs, is often a bottleneck in their development.To provide knowledge on how to improve the in vitrostability of lyophilized ADC formulations, the solid-state stability of ADC formulations with varying risk factors was studied in this dissertation project. The first study investigated interactions between an ADC and excipients in terms of solid-state stability enhancement. The second study investigated the processdriven instability of ADCs during lyophilization using various concentrations of ADCs. The first two studies incorporate a new method called solid-state hydrogen/deuterium exchange coupled with mass spectrometry (ssHDX-MS) as an analytical predictor of solid-state stability. The last study investigated the effects of pH on the stability of labile hydrazones, as a model for common linker chemistry used in ADCs
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