Monoclonal antibodies are large, complex biomolecules that can be difficult to characterize. Characterization is important because of the various post translational modifications that can occur during manufacturing, processing, and storage. Some modifications can lead to efficacy and safety issues and therefore are heavily monitored. A leading way to monitor various modifications is by using liquid chromatography. The high sensitivity, reproducibility, and ability to quantitate analytes makes it very attractive for monoclonal antibody characterization. The large molecular size of monoclonal antibodies (150 kDa) makes them challenging to separate efficiently and with high enough resolution to be helpful. New column technologies that would help improve protein separation efficiencies and slectivities would greatly help in this challenging process. In this thesis, three novel bonded phases are developed for the separation of monoclonal antibodies including a weak anion and cation exchanger (WAX, CEX) for the separation of charged species as well as a novel hydrophilic interaction chromatography (HILIC) for the separation of glycoforms. Column develop is achieved by optimizing selectivity and improving efficiency of separations by altering particle surface chemistry.

Hydrophobic interaction chromatography (HIC) is a common purification method for biological drug substances. For continuous monitoring the quality of the purification, a column performance test is carried out to test the packing of the resin. The packing integrity is commonly tested between runs by measuring the Hight Equivalent to a Theoretical Plate (HETP) by injecting a small volume of a tracer solution (spike) that gives a change in conductivity. The column performance test provides useful information regarding the packing quality that could be used for troubleshooting before a purification is performed since a well packed gel is essential for an effective purification. During this thesis project a screening of suitable test conditions for a HETP test for a HIC column was performed. The developed HETP test were then investigated if it could be combined with the storage of the resin or if the packing is affected by the test conditions. Factors such as spike volume, flow rate and the concentration of the elute and spike solution, were considered. 0.1 M NaOH elute and 0.15 M NaCl as spike solution gave the best result based on HETP and the asymmetry of the conductivity peak. A test purification with the new HETP test conditions compared with the old method were then carried out to verify that the quality of the purification is not affected. The purification showed promising result and implementation of a more convenient storage solution and HETP test would be possible without changing the product quality.

This thesis focuses on the development of new stationary phases for use in hydrophilic interaction liquid chromatography using TRIS-based and phosphorylcholine typed monomers and porous silica particles as starting substrates. In this thesis, several ways of polymerizing highly hydrophilic mono­mers onto pore surfaces of silica supports are described, based on several “grafting from” schemes. “Controlled/living” radical polymerizations including atom transfer radical polymerization (ATRP) and iniferter-mediated polymerization in conjunction with conventional free radical polymerization are demonstrated to be successful tools for grafting different hydrophilic monomers (polyhydroxyl and phosphorylcholine [meth]acrylamide/acrylates) onto the silica surfaces. Reaction solvents are proven to play an essential role to achieve efficient graft polymerization of activated silica surfaces with these amphiphilic vinylic monomers, which is difficult because of their restricted access to the activated surface in solvents that can be used because of solubility constraints. Two tentacle TRIS-based polymer grafted silica, namely TRIS-WAX – TRIS functionality bonded to silica via a C–N–C imine bond and TRIS-Amide – TRIS bonded to silica via an amide bond, prove to be useful as stationary phases for hydrophilic interaction chromatography (HILIC).The TRIS-WAX exhibits a mixed mode hydrophilic partitioning and weak anion exchange (HILIC/WAX) retention mechanism while retention by hydrophilic partitioning is the dominant mechanism on the neutral TRIS-Amide phase which lacks weak anion exchange (WAX) properties. Interestingly, both these phases have selectivities that are radically different from most commercial HILIC stationary phases. Finally, a method is demonstrated for synthesizing a stratified (graft-copolymerized) silica material based on N,N′-methylenebisacrylamide and 2-methacryloyloxyethyl phosphorylcholine (MPC) using a “controlled/living” photoiniferter-mediated polymerization from the N,N-diethyldithiocarbamate iniferter moiety immobilized silica surfaces. This polymerization method proves to be successful for graft-blockcopolymerization of different highly hydrophilic monomers onto the activated surfaces of porous silica. In this way, silica surfaces are grafted with a cross-linked amide-based hydrogel, on top of which a tentacle zwitterionic phosphorylcholine-typed layer is synthesized. The resulted material proves to be useful for HILIC separations and possesses different selectivity for the tested organic acids compared to that of commercial ZIC-cHILIC stationary phase.

<p>The two techniques aqueoustwo-phase partitioning and expanded bed adsorption that bothare suitable for primary protein recovery were studied. Most ofthe work was focused on partition in aqueous two-phase systemsand in particular on the possibility to effect the partitionbehaviour by fusion of short peptide tags or protein domains tothe target protein.</p><p>The partitioning of fusionproteins between different variants of the domain tag Z and thenaturally occurring protein DNA Klenow polymerase were studiedin Breox/Reppal aqueous two-phase systems. Most studies wereperformed with cell homogenate. The Breox/Reppal system was infocus because if the fusion protein can be partitioned to theBreox-rich top phase the next step can be a thermoseparatingaqueous two-phase system. When the Breox phase is heated to50°C it switches from a one-phase system to a two-phasesystem resulting in an almost pure water rich top phase andhighly concentrated Breox-rich bottom phase. The Breox can thenbe reused and the protein recovered from the water phase. TheZ-domain was genetically modified in different ways to Z_basic1, Z_acid2and Z_trp12and fused to the Klenow protein to try toenhance partitioning to the Breox-rich phase. From theexperiments it was not possible to observe any effects on thepartition behaviour irrespectively of tested properties of thedomain tag. Despite the absence of domain tag effects highK-values, i.e. partition to the Breox-rich top phase, wereobserved in the Breox/Reppal system. However, the proteinK-values seemed to be rather sensitive to the cell homogenateload and showed a tendency to decrease with increased cellhomogenate load. Also increased phosphate concentration reducedthe K-values. The partitioning of cell debris also seemed todependent on the cell homogenate load. At higher homogenateload (<=20g DW/L) clear Breox-rich top phases were observedwith the cell debris collected in Reppal-rich bottomphases.</p><p>Two different tetrapeptides,AlaTrpTrpPro and AlaIleIlePro were inserted near the C-terminusof the protein ZZT0. The Trp-rich peptide unit stronglyincreased both the partitioning of ZZT0 into the poly(ethyleneglycol) (PEG)-rich phase in a PEG/potassium phosphate aqueoustwo-phase system and its retention on PEG and propylhydrophobic interaction chromatographic columns with potassiumphosphate as eluent in isocratic systems. Both the partitioningand the retention increased with increasing number of Trp-richpeptide units inserted into ZZT0. Insertion of Ile-richtetrapeptide units affected the partitioning and retention to amuch lesser extent. Partition and modelling data also indicateda folding of inserted Trp and Ile tetrapeptide units, probablyto minimise their water contact. It was also investigated howto predict the partitioning of proteins in isoelectricPEG/phosphate aqueous two-phase systems.</p><p>The capture ofß-galactosidase from<i>E. coli</i>cell homogentate (50g DW/L) by metal chelatexpanded bed adsorption was studied. These experiments showedthat capture, with a certain degree of selectivity, andclarification of ß-galactosidase could be achieved from acell homogenate. However, a rather low recovery of about 35 %was obtained at a capacity of 0.25mg/mL of gel. Thus, severalparameters remain to be optimised like the load buffercomposition and the cell homogenate load.</p><p><b>Keywords:</b><i>E. coli</i>, aqueous two-phase systems, fusion proteins,hydrophobic interaction chromatography, expanded bedadsorption, ß-galactosidase, Klenow polymerase, Z-domain,peptide tags</p>

<p>LC-MS of protein drugs requires new ideas in bonded phase design rather than adapting bonded phases from the realm of small-molecule drugs. The polymer-shell bonded phase is designed to interact with larger molecules and to shield proteins from the silica substrate. The particles consist of a core of solid silica and a shell of dense polymer brush. The polymer layer is thick enough to protect the protein from interactions with silanols to reduce peak tailing. The polymer contains multiple functional groups that introduce more selectivity. This design gives unprecedented LC resolution and MS sensitivity. Our group has developed polymer shell bonded phases for hydrophobic interaction chromatography (HIC-MS) of antibody-drug conjugates (ADCs), hydrophilic interaction liquid chromatography (HILIC-MS) of glycoproteins, and reversed-phase liquid chromatography (RPLC-MS) of monoclonal antibodies. Since HIC is not in-line compatible with MS due to the high salt levels, it is laborious to identify the constituents of HIC peaks. An MS-compatible alternative to HIC is reported here: native reversed phase liquid chromatography (nRPLC). This employs a mobile phase 50 mM ammonium acetate for high sensitivity in MS, and elution with a gradient of water/isopropanol. The nRPLC-MS data show that all ADC species, ranging from drug-to-antibody ratios of 1 to 8, remained intact and native on the column. As we adapt this concept to intact proteins, we find that lysozyme and α-chymotrypsinogen A are both eluted in their native conformations. We also use the polymer-shell concept to resolve IgG1 free thiol variants by RPLC-MS with 0.5% formic acid. Since there are always other variants besides the intended ones, the need for high MS sensitivity is desired to distinguish subtle mass change between disulfide bond and free thiols. Overall, MS sensitivity increases 10X relative while all of the thiol variants are well resolved by the polymethylmethacrylate bonded phase.</p>

As agrarian society developed, the most fertile soils able to sustain the nutritional requirements needed for high crop yield were assigned to farming, while the more penurious soils were left to uphold the forest ecosystems. Some temperate forests are developed on acidic soils considered to be nutrient poor, as much of the inorganic nutrients are entrapped in poorly weatherable soil minerals and not easily accessed by plant roots. In an undisturbed ecosystem, the largest contribution of available nutrients comes from the recycling of organically bound nutrients via the decomposition of dead plant material. If biomass is removed, for instance with a more intensified exploitation of the forest ecosystems including whole tree harvesting, this source of nutrients is consequently decreased. The importance of soil mineral weathering as a source of nutrients, and especially that promoted by soil biota, is thereby emphasized. This thesis addresses biotic parameters associated with mineral weathering. Different aspects of soil solution sampling strategies and analysis of different organic ligands as well as biomarkers for the estimation of fungal biomass were investigated. These chemical parameters were also evaluated as indicators of microbial activity in relation to mineral nutrient availability in soil. With the assumption that the current nutrient status of a soil will affect the microbial interest of certain minerals as sources of inorganic nutrients, a mineral amendment trial was performed in a Swedish boreal forest soil. Overall, the amended soil presented good nutrient status, but with a possible shortage of iron. Due to this, it was hypothesized that the amended mineral with the highest iron content i.e. biotite would cause an elevation of microbial activity in its vicinity when compared to the bulk soil. The level of microbial activity in the vicinity of the amended minerals was evaluated via quantification of organic acids and siderophores, as well as estimation of fungal biomass and enzymatic activity. The highest microbial activity was measured for the O horizon of the investigated podzol, although nothing indicated an elevated association with the amended minerals. In the E horizon, however, elevation in microbial activity was observed in the vicinity of the biotite mineral when compared with bulk soil, although only a few of the investigated parameters differed significantly when evaluated separately.   To enable this study, a highly sensitive analytical method employing liquid chromatography and mass spectrometry was developed to quantify a number of hydroxamate siderophores. On-line pre-concentration enabled detection of these organic ligands in the pico-molar range – a necessity when analyzing natural samples. Furthermore, an analytical method was developed for the estimation of fungal biomass via quantification of chitin-derived glucosamine, which also employed liquid chromatography and tandem mass spectrometry. Unlike currently available methods, the one presented in this thesis did not involve analyte derivatization, which resulted in high sample throughput while simultaneously avoiding complications involved with the additional derivatization procedure. The distribution of a group of organic ligands known as aromatic low molecular mass organic acids was also studied in a boreal forest podzol soil. Different sampling and samples preparation techniques, namely tension-lysimeters, soil centrifugation and liquid-soil extraction, were compared when analyzing soil solution components. Significant differences in analyte amount and species type were found between these sampling techniques. Some of the differences could be accounted for by variation in soil composition at different depths of the investigated podzol, but others could be attributed to structural differences within the studied analyte group. This clearly illustrated the intricacy of sampling and analysis when working with a sample matrix as complex and diverse as soil. As previously, liquid chromatography and mass spectrometry was used to quantify the analytes of interest. A highly sensitive analytical method was developed that was able to detect eleven aromatic low molecular mass organic acids in the nano-molar range. High selectivity was ensured by applying multiple reaction monitoring enabled by collision induced fragmentation of the analytes. / FORE

The two techniques aqueoustwo-phase partitioning and expanded bed adsorption that bothare suitable for primary protein recovery were studied. Most ofthe work was focused on partition in aqueous two-phase systemsand in particular on the possibility to effect the partitionbehaviour by fusion of short peptide tags or protein domains tothe target protein. The partitioning of fusionproteins between different variants of the domain tag Z and thenaturally occurring protein DNA Klenow polymerase were studiedin Breox/Reppal aqueous two-phase systems. Most studies wereperformed with cell homogenate. The Breox/Reppal system was infocus because if the fusion protein can be partitioned to theBreox-rich top phase the next step can be a thermoseparatingaqueous two-phase system. When the Breox phase is heated to50°C it switches from a one-phase system to a two-phasesystem resulting in an almost pure water rich top phase andhighly concentrated Breox-rich bottom phase. The Breox can thenbe reused and the protein recovered from the water phase. TheZ-domain was genetically modified in different ways to Zbasic1, Zacid2and Ztrp12and fused to the Klenow protein to try toenhance partitioning to the Breox-rich phase. From theexperiments it was not possible to observe any effects on thepartition behaviour irrespectively of tested properties of thedomain tag. Despite the absence of domain tag effects highK-values, i.e. partition to the Breox-rich top phase, wereobserved in the Breox/Reppal system. However, the proteinK-values seemed to be rather sensitive to the cell homogenateload and showed a tendency to decrease with increased cellhomogenate load. Also increased phosphate concentration reducedthe K-values. The partitioning of cell debris also seemed todependent on the cell homogenate load. At higher homogenateload (&lt;=20g DW/L) clear Breox-rich top phases were observedwith the cell debris collected in Reppal-rich bottomphases. Two different tetrapeptides,AlaTrpTrpPro and AlaIleIlePro were inserted near the C-terminusof the protein ZZT0. The Trp-rich peptide unit stronglyincreased both the partitioning of ZZT0 into the poly(ethyleneglycol) (PEG)-rich phase in a PEG/potassium phosphate aqueoustwo-phase system and its retention on PEG and propylhydrophobic interaction chromatographic columns with potassiumphosphate as eluent in isocratic systems. Both the partitioningand the retention increased with increasing number of Trp-richpeptide units inserted into ZZT0. Insertion of Ile-richtetrapeptide units affected the partitioning and retention to amuch lesser extent. Partition and modelling data also indicateda folding of inserted Trp and Ile tetrapeptide units, probablyto minimise their water contact. It was also investigated howto predict the partitioning of proteins in isoelectricPEG/phosphate aqueous two-phase systems. The capture ofß-galactosidase fromE. colicell homogentate (50g DW/L) by metal chelatexpanded bed adsorption was studied. These experiments showedthat capture, with a certain degree of selectivity, andclarification of ß-galactosidase could be achieved from acell homogenate. However, a rather low recovery of about 35 %was obtained at a capacity of 0.25mg/mL of gel. Thus, severalparameters remain to be optimised like the load buffercomposition and the cell homogenate load. <b>Keywords:</b>E. coli, aqueous two-phase systems, fusion proteins,hydrophobic interaction chromatography, expanded bedadsorption, ß-galactosidase, Klenow polymerase, Z-domain,peptide tags / NR 20140805

La cellulose et ses dérivés sont utilisés dans un vaste nombre d’applications incluant le domaine pharmaceutique pour la fabrication de médicaments en tant qu’excipient. Différents dérivés cellulosiques tels que le carboxyméthylcellulose (CMC) et l’hydroxyéthylcellulose (HEC) sont disponibles sur le commerce. Le degré de polymérisation et de modification diffèrent énormément d’un fournisseur à l’autre tout dépendamment de l’origine de la cellulose et de leur procédé de dérivation, leur conférant ainsi différentes propriétés physico-chimiques qui leurs sont propres, telles que la viscosité et la solubilité. Notre intérêt est de développer une méthode analytique permettant de distinguer la différence entre deux sources d’un produit CMC ou HEC. L’objectif spécifique de cette étude de maitrise était l’obtention d’un profil cartographique de ces biopolymères complexes et ce, par le développement d’une méthode de digestion enzymatique donnant les oligosaccharides de plus petites tailles et par la séparation de ces oligosaccharides par les méthodes chromatographiques simples. La digestion fut étudiée avec différents paramètres, tel que le milieu de l’hydrolyse, le pH, la température, le temps de digestion et le ratio substrat/enzyme. Une cellulase de Trichoderma reesei ATCC 26921 fut utilisée pour la digestion partielle de nos échantillons de cellulose. Les oligosaccharides ne possédant pas de groupements chromophores ou fluorophores, ils ne peuvent donc être détectés ni par absorbance UV-Vis, ni par fluorescence. Il a donc été question d’élaborer une méthode de marquage des oligosaccharides avec différents agents, tels que l’acide 8-aminopyrène-1,3,6-trisulfonique (APTS), le 3-acétylamino-6-aminoacridine (AA-Ac) et la phénylhydrazine (PHN). Enfin, l’utilisation de l’électrophorèse capillaire et la chromatographie liquide à haute performance a permis la séparation des produits de digestion enzymatique des dérivés de cellulose. Pour chacune de ces méthodes analytiques, plusieurs paramètres de séparation ont été étudiés. / Cellulose and its derivatives are used in a wide range of applications, including the pharmaceutical industry for the manufacturing of medicines as inactive additives. Various cellulosic derivatives such as carboxymethylcellulose (CMC) and hydroxyethylcellulose (HEC) are readily available for such use. The degree of polymerization and modification differs from one supplier to the other, according to the origin of the cellulose and its process of chemical modification, conferring on them different physico-chemical properties, such as viscosity and solubility. Our interest is to develop an analytical method that can distinguish between different sources of a given CMC or HEC product. The specific objective of this master’s study was to obtain a fingerprint of these complex biopolymers by developing an enzymatic digestion method to produce smaller oligosaccharides that could be separated by simple chromatographic methods. The digestion was studied as a function of various parameters, such as the composition of the hydrolysis solution, the pH, the temperature, the duration of digestion and the substrate/enzyme ratio. A cellulase enzyme from Trichoderma reesei ATCC 26921 was used for the partial digestion of our samples of cellulose. Since these oligosaccharides do not possess a chromophore or fluorophore, they can’t be detected either by absorbance or fluorescence. It was thus necessary to work out the labeling method for oligosaccharides using various agents, such as 8-aminopyrene-1,3,6-trisulfonic acid (APTS), 3-acetylamino-6-aminoacridine (AA-Ac) and phenylhydrazine (PHN). Finally, the use of capillary electrophoresis and high performance liquid chromatography allowed the separation of the enzymatic digestion products of the cellulose derivatives (CMC and HEC). For each of these analytical separation techniques, several parameters of the separation were studied.

La cellulose et ses dérivés sont utilisés dans un vaste nombre d’applications incluant le domaine pharmaceutique pour la fabrication de médicaments en tant qu’excipient. Différents dérivés cellulosiques tels que le carboxyméthylcellulose (CMC) et l’hydroxyéthylcellulose (HEC) sont disponibles sur le commerce. Le degré de polymérisation et de modification diffèrent énormément d’un fournisseur à l’autre tout dépendamment de l’origine de la cellulose et de leur procédé de dérivation, leur conférant ainsi différentes propriétés physico-chimiques qui leurs sont propres, telles que la viscosité et la solubilité. Notre intérêt est de développer une méthode analytique permettant de distinguer la différence entre deux sources d’un produit CMC ou HEC. L’objectif spécifique de cette étude de maitrise était l’obtention d’un profil cartographique de ces biopolymères complexes et ce, par le développement d’une méthode de digestion enzymatique donnant les oligosaccharides de plus petites tailles et par la séparation de ces oligosaccharides par les méthodes chromatographiques simples. La digestion fut étudiée avec différents paramètres, tel que le milieu de l’hydrolyse, le pH, la température, le temps de digestion et le ratio substrat/enzyme. Une cellulase de Trichoderma reesei ATCC 26921 fut utilisée pour la digestion partielle de nos échantillons de cellulose. Les oligosaccharides ne possédant pas de groupements chromophores ou fluorophores, ils ne peuvent donc être détectés ni par absorbance UV-Vis, ni par fluorescence. Il a donc été question d’élaborer une méthode de marquage des oligosaccharides avec différents agents, tels que l’acide 8-aminopyrène-1,3,6-trisulfonique (APTS), le 3-acétylamino-6-aminoacridine (AA-Ac) et la phénylhydrazine (PHN). Enfin, l’utilisation de l’électrophorèse capillaire et la chromatographie liquide à haute performance a permis la séparation des produits de digestion enzymatique des dérivés de cellulose. Pour chacune de ces méthodes analytiques, plusieurs paramètres de séparation ont été étudiés. / Cellulose and its derivatives are used in a wide range of applications, including the pharmaceutical industry for the manufacturing of medicines as inactive additives. Various cellulosic derivatives such as carboxymethylcellulose (CMC) and hydroxyethylcellulose (HEC) are readily available for such use. The degree of polymerization and modification differs from one supplier to the other, according to the origin of the cellulose and its process of chemical modification, conferring on them different physico-chemical properties, such as viscosity and solubility. Our interest is to develop an analytical method that can distinguish between different sources of a given CMC or HEC product. The specific objective of this master’s study was to obtain a fingerprint of these complex biopolymers by developing an enzymatic digestion method to produce smaller oligosaccharides that could be separated by simple chromatographic methods. The digestion was studied as a function of various parameters, such as the composition of the hydrolysis solution, the pH, the temperature, the duration of digestion and the substrate/enzyme ratio. A cellulase enzyme from Trichoderma reesei ATCC 26921 was used for the partial digestion of our samples of cellulose. Since these oligosaccharides do not possess a chromophore or fluorophore, they can’t be detected either by absorbance or fluorescence. It was thus necessary to work out the labeling method for oligosaccharides using various agents, such as 8-aminopyrene-1,3,6-trisulfonic acid (APTS), 3-acetylamino-6-aminoacridine (AA-Ac) and phenylhydrazine (PHN). Finally, the use of capillary electrophoresis and high performance liquid chromatography allowed the separation of the enzymatic digestion products of the cellulose derivatives (CMC and HEC). For each of these analytical separation techniques, several parameters of the separation were studied.

Human α-N-Acetylglucosaminidase (Naglu) is a lysosomal acid hydrolase implicated in tthe rare metabolic storage disorder known as mucopolysaccharidosis type IIIB (MPS IIIB; also Sanfilippo syndrome B). Absence of this enzyme results in cytotoxic accumulation of heparan sulphate in the central nervous system, causing mental retardation and a shortened lifespan. Enzyme replacement therapy is not currently effective to treat neurological symptoms due to the inability of exogenous Naglu to access the brain. This laboratory uses a Spodoptera frugiperda (Sf9) insect cell system to express Naglu fused to a synthetic protein transduction domain with the intent to facilitate delivery of Naglu across the blood-brain barrier. The project described herein may be broken down into three main sections. Firstly, the impact of two cryptic splice sites on Naglu expression levels was analyzed in both transiently expressing Sf9 cultures and stably selected cell lines. Secondly, the effectiveness of the native Naglu secretion-signaling peptide in the Sf9 system was examined. Finally, purification of a Naglu fusion protein from suspension culture medium was performed using hydrophobic interaction chromatographic techniques. The ultimate goal of this research is to develop an efficient system for economical, large-scale production of a human recombinant Naglu fusion protein that has the potential to be successfully used for enzyme replacement therapy to treat MPS IIIB. / Graduate