Development of Computer-Aided Molecular Design Methods for Bioengineering Applications

Computer-aided molecular design (CAMD) offers a methodology for rational product design. The CAMD procedure consists of pre-design, design and post-design phases. CAMD was used to address two bioengineering problems: design of excipients for lyophilized protein formulations and design of ionic liquids for use in bioseparations. Protein stability remains a major concern during protein drug development. Lyophilization, or freeze-drying, is often sought to improve chemical stability. However, lyophilization can result in protein aggregation. Excipients, or additives, are included to stabilize proteins in lyophilized formulations. CAMD was used to rationally select or design excipients for lyophilized protein formulations. The use of solvents to aid separation is common in chemical processes. Ionic liquids offer a class of molecules with tunable properties that can be altered to find optimal solvents for a given application. CAMD was used to design ionic liquids for extractive distillation and in situ extractive fermentation processes. The pre-design phase involves experimental data gathering and problem formulation. When available, data was obtained from literature sources. For excipient design, data of percent protein monomer remaining post-lyophilization was measured for a variety of protein-excipient combinations. In problem formulation, the objective was to minimize the difference between the properties of the designed molecule and the target property values. Problem formulations resulted in either mixed-integer linear programs (MILPs) or mixed-integer non-linear programs (MINLPs). The design phase consists of the forward problem and the reverse problem. In the forward problem, linear quantitative structure-property relationships (QSPRs) were developed using connectivity indices. Chiral connectivity indices were used for excipient property models to improve fit and incorporate three-dimensional structural information. Descriptor selection methods were employed to find models that minimized Mallow's Cp statistic, obtaining models with good fit while avoiding overfitting. Cross-validation was performed to access predictive capabilities. Model development was also performed to develop group contribution models and non-linear QSPRs. A UNIFAC model was developed to predict the thermodynamic properties of ionic liquids. In the reverse problem of the design phase, molecules were proposed with optimal property values. Deterministic methods were used to design ionic liquids entrainers for azeotropic distillation. Tabu search, a stochastic optimization method, was applied to both ionic liquid and excipient design to provide novel molecular candidates. Tabu search was also compared to a genetic algorithm for CAMD applications. Tuning was performed using a test case to determine parameter values for both methods. After tuning, both stochastic methods were used with design cases to provide optimal excipient stabilizers for lyophilized protein formulations. Results suggested that the genetic algorithm provided a faster time to solution while the tabu search provides quality solutions more consistently. The post-design phase provides solution analysis and verification. Process simulation was used to evaluate the energy requirements of azeotropic separations using designed ionic liquids. Results demonstrated that less energy was required than processes using conventional entrainers or ionic liquids that were not optimally designed. Molecular simulation was used to guide protein formulation design and may prove to be a useful tool in post-design verification. Finally, prediction intervals were used for properties predicted from linear QSPRs to quantify the prediction error in the CAMD solutions. Overlapping prediction intervals indicate solutions with statistically similar property values. Prediction interval analysis showed that tabu search returns many results with statistically similar property values in the design of carbohydrate glass formers for lyophilized protein formulations. The best solutions from tabu search and the genetic algorithm were shown to be statistically similar for all design cases considered. Overall the CAMD method developed here provides a comprehensive framework for the design of novel molecules for bioengineering approaches

Rational development of protein formulations in solid and solution states

Development of protein formulations in the solid and solution state involves stability studies during long-term storage (2-3 years). A long-term study of a protein under conditions leading to its rapid physical and chemical degradation often results in excessive use of resources and severe time constraints. To minimize these problems, a thorough preformulation study of protein behavior under different conditions is necessary. An in depth understanding of the properties of proteins in both the solution and solid state may subsequently result in selection of conditions leading to adequate stability during storage. Preformulation studies of a protein in solution often involve a three step approach. In this method a protein is first characterized under a range of conditions (e.g. pH, temperature, etc.), and the data is then summarized in the form of an empirical phase diagram. This information is then used to design a high throughput screening approach to identify stabilizing compounds. This approach was employed for preformulation studies of vaccines against Clostridium difficile ( C. difficile)-associated disease. Such vaccines contain formaldehyde treated toxoids A and B in free or adjuvant bound form. Studies of C. difficile toxins and toxoids under a range of conditions revealed a stabilizing effect of formaldehyde crosslinking on the thermal stability of the toxoids. Furthermore, screening for stabilizing compounds resulted in the identification of conditions and specific compounds that lead to enhanced thermal stability of free and bound to adjuvant toxoids. Preformulation studies of proteins in the solid state usually involve characterization of an amorphous solid in general (e.g. moisture content, crystallinity, structural relaxation, etc.) and specific protein properties (e.g. extent of protein structure preservation). Unfortunately, these characteristics of the solid and protein cannot usually predict protein stability during storage. Therefore, a more in depth understanding of amorphous matrices is needed. To understand the role of interactions between protein and expient as well as the homogeneity of protein/excipient mixtures, a study of a model system containing human Growth Hormone (hGH) and sugars (sucrose and trehalose) was performed. This study revealed that the extent of protein/excipient interaction can be used to describe the degree of homogeneity of a lyophilized mixture which can be related to the cryo- and lyo-protecting properties of the excipients. Additionally, it was seen that the rate of structural relaxation is proportional to the rate of insoluble aggregate formation. These studies of proteins in solution and the solid state allowed for the identification of conditions for long term stability studies of C. difficile vaccines and contributed to our understanding of the role of interactions between protein and excipient in lyophilized solids

Chemical and Physical Characterization of Therapeutic Proteins in Solution and Amorphous Solids

The chemical and physical stability of proteins in solution and solids was addressed in this dissertation. Protein-excipient interactions in lyophilized solids were studied by hydrogen/deuterium exchange with mass spectrometry (chapter 3) while glycosylation quanitification (chapter 4) and deamidation (chapter 5) was characterized in antibodies in solution. LC/ESI-MS was the method of choice for all studies. Hydrogen/deuterium exchange study showed that the method can be used to obtain region specific information about protein-excipient interactions in solids. It was demonstrated that exchange protection did not occur uniformly along the backbone of the protein and was dependant on excipient type and protein structure. The glycosylation quanitification study demonstrated that the Fc/2 (limited proteolysis followed by reduction) method was relatively quick and accurate and showed comparable values to the standard sugar release assay. Antibody deamidation study demonstrated that secondary structure played a pivotal role in determination of the deamidation products in antibodies

High Resolution Mass Spectrometric Approaches To Study Protein Structure and Environment in Lyophilized Solids

Proteins 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

The influence of moisture content and temperature on storage stability of freeze-dried biologics

Moisture and temperature are both critical factors that affect the long term storage stability of Freeze-dried (FD) biologics. Both physical structure and biological activity can be affected by the conditions that the FD material is subject to over a prolonged storage period. This project aimed to investigate how these key factors affected the long term storage stability of FD biologics. The relationship between moisture content, cake structure and the physical/biological stability for model proteins/antigen standards during long term storage was investigated. Novel techniques and procedures were developed to measure the effects of moisture and storage temperature in FD material. Dynamic vapour sorption (DVS) instrumentation, in conjunction with a real time video imaging, was used to measured visible collapse/shrinkage of FD materials. DVS data used in conjunction with video images for a series of temperatures were analysed to provide stability maps. These provided critical moisture content levels that should not be exceeded in order to retain structural cake stability. In addition, other novel techniques were utilised to measure morphological or physical changes with regards to moisture and temperature. Mechanical properties of the FD materials was measured with a flat punch indenter, while inverse gas chromatography (IGC) was used to measure the cake specific surface area (SSA). Mechanical indention data showed that increasing moisture and storage temperature lead to a reduction of mechanical properties and specifically Young’s modulus. IGC was shown to be suitable alternative to measure SSA of FD biologics, providing comparable SSA values to standard volumetric gas adsorption techniques. Advantages of IGC included being able to show and measure changes to SSA of FD materials conditioned at different relative humidities. A series of long term stability trials were also conducted for high protein concentration formulations (IgG) with a range of 10 - 200 mg/mL to further investigate mechanisms of protein stabilisation in regards to optimum moisture and temperature. Higher concentration proteins had lower SSA’s with larger Young’s Modulus but suffered from longer reconstitution times. IgG stability during 12 month storage trials showed evidence for both vitrification and water replacement theories. The data also provided further evidence for the bell shaped distribution theory of optimum moisture content for some materials and that over-drying with a low moisture cycle might not necessarily be the best option for long term storage stability of IgG. High moisture contents of up to 5% w/w did not seem to have any impact on stability until storage above 45°C. With high concertation FD proteins above 50 mg/mL, there was low of risk of structural collapse with increasing moisture content compared to lower concentration materials. Water ingress into vials during their long term storage is of huge concern especially for all FD materials, but especially so for low mass products such as FD Influenza antigens. Comparison of different closure storage formats for FD antigens was explored and it was found that vials with vacuum-oven dried stoppers had less moisture ingress than vials with unprocessed stoppers (straight out of manufacturers packaging). Vials with vacuum-oven dried stoppers were shown to give comparable potency and moisture content ingress as glass ampoules for reference standard influenza antigens over a 1 year period from -20°C to 45°C. Thus not only can vials with vacuum oven dried stoppers reduce moisture ingress compared to unprocessed stopper vials, but can also result in retaining greater potency and stability during long term storage. In summary, this thesis via use of prolonged stability trials, expanded and further consolidated knowledge on current theories of mechanisms of stability in context to moisture content and temperature during long term storage for real world commercial FD biological standards. This thesis also promoted and endorsed the adoption of novel techniques or practices (such as vacuum oven drying stoppers) to provide further aid and insight in optimising the long term storage stability of FD biologics for future use in industry.Open Acces

Characterization, Stabilization and Formulation Design of IgG and Secretory IgA Monoclonal Antibody Candidates during Storage and Administration

ABSTRACT Monoclonal antibodies (mAbs) have become a class of therapeutic protein-based drugs of high importance for treating numerous human diseases. As a complex, delicate three dimensional molecule, a mAb can be sensitive to ambient environments and thus often display colloidal/conformational instability during manufacturing, storage, and administration. The use of formulation strategies, such as employing specific excipients and/or particular solution conditions (e.g., pH and buffers), can significantly improve a mAb’s pharmaceutical stability properties. In this Ph.D. thesis research work, both formulation/storage stability as well as stability during in vitro models of mAb administration in vivo are evaluated with two different of classes of immunoglobulin molecules (IgG and IgA). In addition, the effect of specific classes of excipients and solution conditions are examined by using a wide variety of physicochemical and immunological binding analytical techniques. Specifically, the second and third chapters of the Ph.D. thesis work focus on the formulation development of high-concentration mAb dosage forms for subcutaneous (SC) injections. Reversible self-association (RSA) of mAbs, which is primarily due to intermolecular protein-protein interactions (PPIs) between mAb molecules, has emerged as an important formulation challenge in terms of significantly increasing solution viscosity and turbidity as well as initiating phase separation. In these two chapters, two different human IgG1 molecules (mAb-J and mAb-C), which showed strong RSA propensity at relatively high protein concentrations, were comprehensively studied to better understand their solution properties and molecular behaviors by both biophysical techniques as well as hydrogen-deuterium exchange mass spectrometry (HX-MS). The aim is to not only characterize mAb molecular properties and solution behavior at relatively high protein concentrations, but also to develop a better mechanistic understanding by identifying peptide segments within the mAb involved PPIs in solution. In these two studies, both elevated solution viscosity and turbidity as well as reduced relative solubility and increased protein-protein interaction propensity (as measured by light scattering profiles and observations of phase separation) were determined for two different mAbs (mAb-J and mAb-C) at comparatively high protein concentrations. Concomitantly, based on the amino acid sequence of each mAb’s RSA sites (as determined by lyophilization-reconstitution-based HX-MS methodology), two different dominant non-covalent forces (electrostatic and hydrophobic) are proposed to be the major driving force for PPIs of the two different mAbs (consistent with previous results). More importantly for this work, varying effects of different excipients were investigated particularly for their ability of promote or disrupt PPIs of each mAb. For mAb-J (electrostatic driven RSA), selected ionic excipients showed the ability to disrupt liquid-liquid phase separation and reduce intermolecular interactions to varying extents, with arginine hydrochloride possessing the highest efficiency. For mAb-C (hydrophobic driven RSA), opposing effects were observed for sodium sulfate versus selected hydrophobic additives (e.g., specific salts, amino acids, solvents), showing both enhanced and reduced PPI propensity, respectively. In both studies, not only was the RSA of mAbs shown to be mAb concentration dependent, but the excipient’s ability to mitigate the RSA of mAbs RSA also displayed an excipient concentration dependent pattern. The fourth chapter focuses on the possibility of using various classes of mAbs, including secretory IgA (sIgA) and IgG1, as potential drug candidates for oral delivery to prevent enteric diseases in infants. Specifically, the use of mAbs against enterotoxigenic Escherichia coli (ETEC) is examined with the idea that passive immunization by pathogen-specific immunoglobulins, by oral delivery to infants, is promising approach to provide “instant” protection against ETEC. Secretory IgA (sIgA) is of particular interest because it is naturally found in the mucosal surfaces within the GI tract, is relatively more resistant to proteolysis by digestive enzymes (vs. IgG), and can protect against enteric bacteria by directly neutralizing virulence factors. In this study, three different mAbs, (sIgA1, sIgA2 and IgG1) against heat labile toxin (LT, one of the major virulence factors of ETEC), were used as a model for developing analytical techniques to characterize the structural integrity of the mAbs and to assess their stability profiles under various solution conditions (using physicochemical and immunochemical binding assays). In this work, very different total carbohydrate levels and N-linked glycosylation oligosaccharide composition profiles were observed between sIgAs and IgG1 made from CHO cell lines. According to SDS-PAGE, SE-HPLC, and SV-AUC results, heterogeneous mixtures of higher molecular weight species were observed for sIgAs, while IgG1 samples showed less heterogeneity with more than 90% monomer in solution. The overall physical stability results at both pH 7.2 and pH 3.0 demonstrated that both sIgA1 and sIgA2 were more stable than IgG1, with sIgA1 displaying the best stability profile. The relative solubility profile of each molecule was pH dependent with higher solubility noted at the lower pH. Furthermore, an in vitro digestion model was adapted in the laboratory to mimic in vivo oral gastric degradation conditions using minimal material, and was utilized to monitor the oral delivery stability of the three mAbs. It was shown that F(ab’)2 was the major digestion product by pepsin digestion. Both sIgAs displayed better resistance to degradation by proteases at low pH compared to IgG1. Moreover, the sIgAs showed greater retention of LT-antigen binding activity than that of IgG1, confirming the superior pharmaceutical properties of sIgAs for oral delivery. In summary, we hope to use the information gained by these preformulation characterization studies for the long-term goal to design stable, low-cost liquid formulations for oral delivery of sIgAs to protect against enteric diseases currently affecting infants in the developing world

Nanofibrillar cellulose for encapsulation and release of pharmaceuticals

The main role of excipients is to ensure the safety and efficacy of the whole pharmaceutical formulation throughout its shelf-life and administration. Formulation design and development as well as material testing are the key components for successful drug delivery. This is becoming increasingly complicated as new active pharmaceutical ingredients typically have poor solubility and/or bioavailability. Due to this, there is an ever increasing need to explore new excipients and material combinations as innovative formulation solutions are required. Furthermore, modified release formulations are needed to control the release rates and to adjust the desired therapeutic effects, raising even more demand for effective formulations. The main aim of this thesis was to evaluate the performance of plant based materials nanofibrillar cellulose (NFC) and anionic carboxylated nanofibrillar cellulose (ANFC) as pharmaceutical excipients for modified release formulations and bioadhesive films. These materials are widely available from renewable sources; biocompatible with relatively low toxicity combined with high mechanical strength and large surface area available for encapsulation. NFC and ANFC, together with HFBII protein, were used as emulsion stabilizers for encapsulation and release of poorly water-soluble drugs. The synergistic stabilization mechanism achieved with these biopolymers improved emulsions stability with extremely low concentrations. In another study, ANFC hydrogels were evaluated as matrix reservoirs for diffusion controlled drug release. Their rheological and drug release properties were shown to be preserved after freeze-drying and reconstruction. The ANFC hydrogels controlled the release kinetics of small molecular weight drugs moderately, whereas significant control was obtained in the case of large proteins. In a comparative study, three new grades of microcrystalline cellulose (MCC) hydrogels were evaluated for diffusion controlled drug release. MCC matrices efficiently controlled the release of both large and small compounds, indicating great potential for drug release applications in a similar manner to the ANFC hydrogels. Bioadhesive NFC and ANFC based films were prepared by incorporating bioadhesive polymers mucin, pectin and chitosan into the film structure. The bioadhesive properties of the films combined with good mechanical and hydration properties, together with low toxicity makes them a feasible option for buccal drug delivery applications. In conclusion, NFC and ANFC were shown to be versatile excipients applicable for several types of dosage forms. In the future, it is seen that these materials may be used systematically as functional excipients for modified release dosage form.Apuaineiden tärkein tehtävä on varmistaa farmaseuttisen formulaation turvallisuus ja tehokkuus säilytyksestä annosteluun asti. Formulaation suunnittelu ja kehittäminen sekä materiaalien testaus ovat keskeisiä tekijöitä onnistuneessa lääkkeen annostelussa. Uudet farmaseuttiset yhdisteet ovat kuitenkin tyypillisesti hyvin niukkaliukoisia ja/tai niiden biologinen hyötyosuus on huono, mikä hankaloittaa formulointia. Tästä syystä uusia apuaineita ja materiaaliyhdisteitä on tutkittava, jotta löydettäisiin innovatiivisia ratkaisuja formulointiongelmiin. Lisäksi formulaatioilla voidaan säädellä vapautumisnopeutta ja terapeuttisia vaikutuksia, joka entisestään lisää tehokkaiden formulaatioiden kysyntää. Tämän tutkimuksen tavoitteena oli arvioida kasviperäisten materiaalien, nanofibrilloidun selluloosan (NFC) ja anionisen karboksyloidun nanofibrilloidun selluloosan (ANFC) suorituskykyä farmaseuttisina apuaineina säätövalmisteissa ja bioadhesiivisissa kalvoissa. Nämä materiaalit ovat bioyhteensopivia, helposti saatavilla uusiutuvista luonnonlähteistä ja niiden mekaaninen lujuus sekä pinta-ala ovat suuret. Lisäksi NFC ja ANFC eivät ole toksisia, joten niitä voidaan hyödyntää lääkeaineiden kapseloinnissa. HFBII proteiinia käytettiin NFC:n ja ANFC:n kanssa emulsio-stabilisaattoreina niukkaliukoisten lääkeaineiden kapseloinnissa ja vapauttamisessa. Näiden biopolymeerien synergistinen vaikutus tehosti emulsioiden stabiilisuutta jo erittäin alhaisilla pitoisuuksilla. Toisessa tutkimuksessa, ANFC:n vaikutusta arvioitiin lääkeaineiden varastoitumista ja diffundoitumista hydrogeelimatriisissa. ANFC hydrogeeli sääteli pienten molekyylien vapautumiskinetiikkaa kohtuullisesti, kun taas suurten proteiinien vapautumista merkittävästi. Lisäksi kylmäkuivattujen hydrogeelien reologiset ominaisuudet voitiin säilyttää uudelleen hydratoimisen jälkeen. Kolmannessa tutkimuksessa verrattiin kolmea uutta eri mikrokiteistä selluloosalaatua (MCC) ja niiden kykyä säädellä lääkeaineiden vapautumista. MCC matriisien todettiin säätelevän tehokkaasti sekä pienien että suurien molekyylien vapautumista. Näiden tutkimusten perusteella sekä ANFC että uudet MCC:t ovat potentiaalisia materiaaleja säätövalmisteiden formuloinnissa. Lisäksi tässä työssä valmistettiin bioadhesiivisia NFC ja ANFC kalvoja, yhdistämällä niihin bioadhesiivisia polymeerejä (pektiini, musiini ja kitosaani). Bioadhesiivisten kalvojen mekaaniset ominaisuudet, bioyhteensopivuus, bioadhesiivisuus ja hydrataatio-ominaisuudet mahdollistavat niiden käytön bukkaalisina lääkevalmisteina. NFC ja ANFC toimivat erittäin monipuolisina apuaineina useille eri lääkemuodoille. Tulevaisuudessa näitä materiaalija voidaan käyttää systemaattisesti funktionaalisina apuaineina säätövalmisteissa