Master of Science / Department of Physics / Jeremy D. Schmit / Antibodies are large Y-shaped proteins which are used by immune system to identify and neutralize pathogens. Monoclonal antibody therapy is used to treat different patient conditions. There are problems associated with the manufacturability and deliverability of mAb solutions due to the viscous nature of the protein. The viscosity of antibody solutions increases with the increase in concentration and decreases with applied shear. We want to know why these behaviours are seen and to address this problem we have developed a theory describing the rapid viscosity increase with increasing concentration. We use the polymer theory to explain this behaviour. Here antibodies are treated as polymers. The length of the polymer depend on the aggregation. The reptation time increases approximately as the cubic power of size of aggregate (N³ ). We see the shear thinning behaviour is dependent on the Ab-Ab binding energy and find the relationship between the size of the aggregate and the binding energy. We find aggregate size and morphology using several models for Ab-Ab interaction sites. We use the head to head binding (fAb-fAb binding) model to describe aggregation state in our viscosity theory. The size of the aggregate and hence the reptation time is captured by the binding energy. When the binding energy increases the zero shear viscosity increases and the reptation time decreases. Likewise when the binding energy decreases the zero shear viscosity decreases and the reptation time increases. We have yet to find the correct exponents for the shear thinning behaviour of different mAbs which would be our future work.

Characterisation of phytochrome using monoclonal antibodies Mary L. Holdsworth Native oat phytochrome has been purified to homogeneity and used to produce a panel of monoclonal antibodies (mAbs). Selection of mAbs followed early screening against native phytochrome by ELISA, and SDS-denatured phytochrome by "mini" western blotting. Six mAbs which recognised SDS-denatured phytochrome were mapped using proteolytically derived fragments of phytochrome and subsequent immunoblotting. LAS 31 and 33 map to the 6 kDa NH2-terminus and LAS 35 and 41 map to the adjacent 4 kDa sub-NH2- terminal domain. LAS 11 maps to the 64 kDa- chromophore-bearing domain and LAS 32 maps to between 74 and 88 kDa from the NH2-terminus on the COOH- terminal-half of the molecule. A novel protocol for the mapping of conformation-specific mAbs has been developed and used to assign LAS 21, 34 and 42 to the 64 kDa-chromophore-bearing domain. Determination of differential affinities towards Pr and Pfr demonstrated that LAS 42 exhibited a higher affinity for Pfr, LAS 31, 33, 34 and 35 exhibited a higher affinity for Pr. LAS 41 discriminates absolutely against Pfr. LAS 41 has therefore facilitated:- (i) the purification of PfrP, i.e. Pfr which is free of contaminating Pr, (ii) the development of an ELISA for phytochrome photoequilibrium, (iii) the first direct experimental evidence that phytochrome can exist as a stable heterodimer in vitro and (iv) an appraisal of ELISA protocols for determining differential affinities of mAbs towards Pr and Pfr. Spectral analyses of phytochrome in the presence of mAbs have underlined the importance of the 6 kDa NH2-terminus in the maintenance of the spectral integrity of the molecule but have also indicated that the 4 kDa sub-NH2-terminal domain also interacts with the chromophore. Cross reactivity studies amongst phytochrome from monocots and dicots demonstrate that the epitopes recognised by LAS 11, 31, 33, 35 and 41 are not highly conserved. However, LAS 32 recognises phytochrome from every plant species tested, and is therefore recognising a highly conserved region on the molecule.

Student Number : 9000987E - PhD thesis - School of Medicine - Faculty of Health Sciences / The generation of human monoclonal antibodies (mAbs) that are able to block HIV-1 infection in vitro would be useful reagents for studying virus neutralization, and assist in identifying neutralizing antibody (NAb) epitopes of HIV-1 envelope glycoprotein. This may provide important information for designing HIV-1 vaccine that aim to induce NAbs. HIV-1 subtype C individuals with high levels of NAb titres were identified, and peripheral blood mononuclear cells (PBMC) from these individuals were isolated and B-cells transformed with Epstein-Barr virus (EBV). Clones specific to HIV-1 gp120 using cell lysate preparations derived from HIV-1 subtype C infected cell lines were generated by performing limiting dilutions. Transformation efficiencies were estimated at over 80% by evaluating EBV-transformation cultures by microscopic visualization. Of these approximately 5% were HIV-1-specific. Five clones derived from the Du23 (1) sample secreting anti-HIV-1 antibodies were generated: 2.3C, 2.9D, 3.2C, 4.12E, and 1.5D. The 1.5D mAb could not be confirmed as anti-HIV-1 clone and it was probably lost during the process of subculturing. The remaining four Du23 mAbs were determined to be of IgG1 isotype lambda (λ) light chain. These mAbs bind to gp120, and 2.9D is probably a polyreactive clone. Clones 2.3C, 3.2C and 4.12E appear to be A32-like, but do not share the same epitope. We have determined that the binding sites for all four Du23 mAbs require at least the C1 region, and they also showed binding sites overlapping with F91 and 1.5E. All four Du23 mAbs required intact gp120 proteins for their binding, and soluble CD4 enhance their binding. Thus, their binding site is discontinuous and conformational. These mAbs are non-neutralizing as they showed limited activity of 30-59% when tested using T-cell line grown viruses or 0-30% when tested against pseudovirions. This activity is rather low when compared to over 80% shown by broadly neutralizing mAbs that have been described in the literature. The challenge in generating mAbs, in particular subtype C-derived, is to find those antibodies capable of suppressing viral replication in vivo and be capable of preventing infection. These reagents could be used to identify epitopes to guiding the design of HIV-1 subtype C envelope immunogens or vaccines. It is also envisaged that neutralizing antibodies used in therapeutic setting or in combination with antiviral drug therapy could reduce viral load and retard disease progression in infected people.

This research investigates approaches to improve the performance of multi-agent based simulation (MABS) applications executed in distributed computing environments. MABS is a type of micro-level simulation used to study dynamic systems consisting of interacting entities, and in some cases, the number of the simulated entities can be very large. Most of the existing publicly available MABS tools are single-threaded desktop applications that are not suited for distributed execution. For this reason, general-purpose multi-agent platforms with multi-threading support are sometimes used for deploying MABS on distributed resources. However, these platforms do not scale well for large simulations due to huge communication overheads. In this research, different strategies to deploy large scale MABS in distributed environments are explored, e.g., tuning existing multi-agent platforms, porting single-threaded MABS tools to distributed environment, and implementing a service oriented architecture (SOA) deployment model. Although the factors affecting the performance of distributed applications are well known, the relative significance of the factors is dependent on the architecture of the application and the behaviour of the execution environment. We developed mathematical performance models to understand the influence of these factors and, to analyze the execution characteristics of MABS. These performance models are then used to formulate algorithms for resource management and application tuning decisions. The most important performance improvement solutions achieved in this thesis include: predictive estimation of optimal resource requirements, heuristics for generation of agent reallocation to reduce communication overhead and, an optimistic synchronization algorithm to minimize time management overhead. Additional application tuning techniques such as agent directory caching and message aggregations for fine-grained simulations are also proposed. These solutions were experimentally validated in different types of distributed computing environments. Another contribution of this research is that all improvement measures proposed in this work are implemented on the application level. It is often the case that the improvement measures should not affect the configuration of the computing and communication resources on which the application runs. Such application level optimizations are useful for application developers and users who have limited access to remote resources and lack authorization to carry out resource level optimizations.

No description available.

La simulation multi-agent représente une solution pertinente pour l’ingénierie et l’étude des systèmes complexes dans de nombreux domaines (vie artificielle, biologie, économie, etc.). Cependant, elle requiert parfois énormément de ressources de calcul, ce qui représente un verrou technologique majeur qui restreint les possibilités d'étude des modèles envisagés (passage à l’échelle, expressivité des modèles proposés, interaction temps réel, etc.).Parmi les technologies disponibles pour faire du calcul intensif (High Performance Computing, HPC), le GPGPU (General-Purpose computing on Graphics Processing Units) consiste à utiliser les architectures massivement parallèles des cartes graphiques (GPU) comme accélérateur de calcul. Cependant, alors que de nombreux domaines bénéficient des performances du GPGPU (météorologie, calculs d’aérodynamique, modélisation moléculaire, finance, etc.), celui-ci est peu utilisé dans le cadre de la simulation multi-agent. En fait, le GPGPU s'accompagne d’un contexte de développement très spécifique qui nécessite une transformation profonde et non triviale des modèles multi-agents. Ainsi, malgré l'existence de travaux pionniers qui démontrent l'intérêt du GPGPU, cette difficulté explique le faible engouement de la communauté multi-agent pour le GPGPU.Dans cette thèse, nous montrons que, parmi les travaux qui visent à faciliter l'usage du GPGPU dans un contexte agent, la plupart le font au travers d’une utilisation transparente de cette technologie. Cependant, cette approche nécessite d’abstraire un certain nombre de parties du modèle, ce qui limite fortement le champ d’application des solutions proposées. Pour pallier ce problème, et au contraire des solutions existantes, nous proposons d'utiliser une approche hybride (l'exécution de la simulation est partagée entre le processeur et la carte graphique) qui met l'accent sur l'accessibilité et la réutilisabilité grâce à une modélisation qui permet une utilisation directe et facilitée de la programmation GPU. Plus précisément, cette approche se base sur un principe de conception, appelé délégation GPU des perceptions agents, qui consiste à réifier une partie des calculs effectués dans le comportement des agents dans de nouvelles structures (e.g. dans l’environnement). Ceci afin de répartir la complexité du code et de modulariser son implémentation. L'étude de ce principe ainsi que les différentes expérimentations réalisées montre l'intérêt de cette approche tant du point de vue conceptuel que du point de vue des performances. C'est pourquoi nous proposons de généraliser cette approche sous la forme d'une méthodologie de modélisation et d'implémentation de simulations multi-agents spécifiquement adaptée à l'utilisation des architectures massivement parallèles. / Multi-Agent Based Simulations (MABS) represents a relevant solution for the engineering and the study of complex systems in numerous domains (artificial life, biology, economy, etc.). However, MABS sometimes require a lot of computational resources, which is a major constraint that restricts the possibilities of study for the considered models (scalability, real-time interaction, etc.).Among the available technologies for HPC (High Performance Computing), the GPGPU (General-Purpose computing on Graphics Processing Units) proposes to use the massively parallel architectures of graphics cards as computing accelerator. However, while many areas benefit from GPGPU performances (meteorology, molecular dynamics, finance, etc.). Multi-Agent Systems (MAS) and especially MABS hardly enjoy the benefits of this technology: GPGPU is very little used and only few works are interested in it. In fact, the GPGPU comes along with a very specific development context which requires a deep and not trivial transformation process for multi-agents models. So, despite the existence of works that demonstrate the interest of GPGPU, this difficulty explains the low popularity of GPGPU in the MAS community.In this thesis, we show that among the works which aim to ease the use of GPGPU in an agent context, most of them do it through a transparent use of this technology. However, this approach requires to abstract some parts of the models, what greatly limits the scope of the proposed solutions. To handle this issue, and in contrast to existing solutions, we propose to use a nhybrid approach (the execution of the simulation is shared between both the processor and graphics card) that focuses on accessibility and reusability through a modeling process that allows to use directly GPU programming while simplifying its use. More specifically, this approach is based on a design principle, called GPU delegation of agent perceptions, consists in making a clear separation between the agent behaviors, managed by the processor, and environmental dynamics, handled by the graphics card. So, one major idea underlying this principle is to identify agent computations which can be transformed in new structures (e.g. in the environment) in order to distribute the complexity of the code and modulate its implementation. The study of this principle and the different experiments conducted show the advantages of this approach from both a conceptual and performances point of view. Therefore, we propose to generalize this approach and define a comprehensive methodology relying on GPU delegation specifically adapted to the use of massively parallel architectures for MABS.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Apart from its primary function in the synaptic hydrolysis of acetylcholine, acetylcholinesterase (AChE) has been shown through in vitro demonstrations to be able to promote various non-cholinergic functions, including cell adhesion and neurite outgrowth, differentiation, and amyloidosis. AChE was also shown to bind to mouse laminin-111 in vitro by an electrostatic mechanism. Previous results suggest that the site on AChE recognised by certain monoclonal antibodies (MAbs) might be critical for differentiation. These MAbs were found to inhibit both laminin binding and cell adhesion in neuroblastoma cells. In this study, the structure and characteristics of this site were investigated, using the AChE-laminin interaction as a template as well as a detailed epitope analysis of the MAbs. The interaction sites of AChE and laminin were investigated using phage display, modelling and docking, synthetic peptides, enzyme linked immunosorbent assays (ELISAs) and conformational interaction site mapping. Docking of AChE with the single-chain variable fragments (scFvs) produced from the phage display showed the major recognition motifs to be the 90Arg-Glu-Leu-Ser-Glu-Asp motif, the 40Pro-Pro-Met-Gly sequence, and the 59Val-Val-Asp-Ala-Thr-Thr (human) motif. Mouse AChE was found to interact with the basic structures Val2718-Arg-Lys-Arg- Leu2722; Tyr2738-Tyr2739, Tyr2789-Ile-Lys-Arg-Lys2793; and Val2817-Glu-Arg-Lys2820, on the 1 G4 domain of laminin. ELISAs using synthetic peptides confirmed the involvement of the AG-73 site (2719-2729). This site overlaps with laminin’s heparin-binding site. Docking showed the major component of the interaction site on AChE to be the acidic Arg90-Glu-Leu-Ser-Glu-Asp95 (omega loop), and also involving the Pro40-Pro-Val42, Arg46 (linked to Glu94 by a salt bridge) and the hexapeptide Asp61 Ala-Thr-Thr-Phe-Gln66. Epitope analysis showed the MAb’s major recognition site to be the sequence Pro40-Pro- Met-Gly-Pro-Arg-Arg-Phe48 (human AChE). The MAbs also reacted with the prolinerich sequences Pro78-Gly-Phe-Glu-Gly-Thr-Glu84 and Pro88-Asn-Arg-Glu-Leu-Ser-Glu- Asp95. These results define the interaction sites involved in the AChE-laminin interaction and suggest that the interaction plays a role in cell adhesion. Despite the in vitro demonstrations of the importance of AChE’s non-classical functions, the AChE knockout survives. Results from this study suggest the possibility of functional redundancy between AChE and other molecules in early development. Using these in vitro findings that AChE is able to bind laminin-111, information on the interaction sites, as well as results from the monoclonal antibody (MAb) epitope analysis, the idea of redundancy was investigated. Docking and bioinformatics techniques were used to investigate structurally similar molecules that have comparable spatiotemporal expression patterns in the embryonic nervous system. AChE has been shown to be involved in the pathogenesis of Alzheimer’s disease, thus molecules associated with brain function and neurodegeneration were also investigated. Molecules with which AChE could be possibly redundant are syndecans, glypicans, perlecan, neuroligins and the low-density lipoprotein receptors and their variants. AChE was observed to dock with growth arrest-specific protein 6 (Gas6) as well as apolipoprotein E3 (ApoE-3) at the same site as the laminin interaction. The AChE interaction site was shown to resemble the apolipoprotein-binding site on the low density lipoprotein receptor, and related molecules, including the low density lipoprotein receptor-related molecule (LRP) and the sortilin-related receptor (SORL1). These molecules, along with apoE, are associated with Alzheimer’s disease. Resemblances to the triggering receptor on myeloid cells (TREM1) were also suggested; this is interesting as AChE has been implicated in both haematopoiesis and haematopoietic cancers. Coimmunoprecipitation results, applied to investigate alternative ligands for AChE, confirmed the AChE-laminin interaction in neuroblastoma cells, and also suggested the existence of other binding partners. In conclusion, characterisation of the AChE-laminin interaction sites and investigation of structurally similar sites in other molecules suggests a role for AChE in the stabilization of the basement membrane of developing neural cells and provides a feasible explanation for the survival of the knockout mouse. Furthermore, the demonstrated similarity of the AChE interaction site to sites on molecules, notably the low density lipoprotein receptor family and SORL1 and their apolipoprotein ligands that are implicated in the pathology of Alzheimer’s disease, as well as the possible link to haematopoietic differentiation and cancers, warrants further investigation. / AFRIKAANSE OPSOMMING: Talle in vitro studies wys dat die ensiem asetielcholienesterase (AChE), behalwe vir sy klassieke rol in die hidrolise van asetielcholien (ACh), ‘n aantal nie-cholinerge rolle vertolk insluitend in sel adhesie, in die uitgroei van neurieten, in differensiering, asook in amyloidosis. Dit is vooraf gewys dat AChE, met behulp van elektrostatiese meganismes, in vitro met muis laminin-111 kan bind. Dit word verneem dat die area op AChE wat herken word deur monoklonale teenliggaampies (MAbs), moontlik ‘n kritiese area is met betrekking tot differensiasie. Dieselfde MAbs is gevind om beide die laminin-interaksie, sowel as sel adhesie van neuroblastoma selle, te inhibeer. In hierdie projek word die struktuur en eienskappe van die betrokke kritiese areas ondersoek deur die AChE-laminin interaksie te gebruik as sjabloon. ‘n Gedetailleerde analise van die teenliggaam epitoop het ook geskied. Met behulp van faag vertoon, modellering en hegting, sintetiese peptiede, ensiem-gekoppelde immunosorbent toetse (ELISAs) en konformasie interaksie area kartering, is die betrokke interaksie areas bestudeer. Hegting van enkel-ketting varierende fragment (scFv) volgordes, verkry vanaf die vaag vertoon, aan AChE dui dat die hoof herkennings motiewe die 90Arg-Glu-Leu-Ser-Glu-Asp motief, die 40Pro-Pro- Met-Gly volgorde, en die 59Val-Val-Asp-Ala-Thr-Thr (mens) motief is. ‘n Interaksie tussen muis AChE en die 1 G4 domein van laminin is gevind. Die interaksie betrek die basiese structure: Val2718-Arg-Lys-Arg-Leu2722; Tyr2738-Tyr2739, Tyr2789-Ile-Lys-Arg- Lys2793; en Val2817-Glu-Arg-Lys2820. Die betrokkenheid van die AG-73 (2719-2729) area by hierdie interaksie is bevestig met ELISA eksperimente wat sintetiese peptiede inkorporeer. Die AG-73 area oorvleuel die heparin interaksie area op laminin. Hegtings eksperimente wys dat die hoof komponent van die interaksie area op AChE die suur volgorde Arg90-Glu-Leu-Ser-Glu-Asp95 op die omega-lus is. Die interaksie betrek ook die Pro40-Pro-Val42, Arg46 (gekoppel aan Glu94 deur ‘n sout-brug) en die heksapeptied Asp61 Ala-Thr-Thr-Phe-Gln66 motiewe. Analise van die MAb epitoop wys die hoof erkennings area as volgorde Pro40-Pro-Met-Gly-Pro-Arg-Arg-Phe48 (mens AChE). Die MAbs blyk ook gunstig te wees teenoor prolien-ryke volgordes soos Pro78-Gly-Phe-Glu-Gly-Thr-Glu84 en Pro88-Asn-Arg-Glu-Leu-Ser-Glu-Asp95. Die areas betrokke by die AChElaminin interaksie is dus gedefinieer en ‘n moontlike rol vir hierdie interaksie in sel adhesie word voorgestel. Die noodsaaklikheid van AChE se nie-klassieke funksies word bevraagteken na die oorlewing van die AChE uitklop-muis. Resultate hier dui op die moontlikheid van funksionele oortolligheid as verduideliking hiervan, spesifiek met betrekking tot molekules betrokke in vroëe ontwikkeling asook in die proses van neurale agteruitgang. Deur gebruik te maak van die in vitro demonstrasies van die AChE-laminin interaksie, informasie verkry ten opsigte van die betrokke interaksie areas, asook resultate verkry vanaf die monoklonale teenliggaam (MAb) epitoop analise, word die idee van funksionele oortolligheid ondersoek. Hegtings en bioinformatika tegnieke is gebruik om molekules met soortgelyke strukture en uitdrukkings patrone in die embrioniese senuweestelses te ondersoek. Ko-immuno presipitasie tegnieke is gebruik om so moontlike alternatiewe ligande vir AChE te ondersoek. Moontlike funksionele oortolligheid van AChE met die volgende molekules is gevind: syndecan; glypican; perlecan; neuroligin; asook die lae-digtheid lipoproteien (LDL) reseptore en hul variante. Hegting van AChE met ’growth arrest-specific’ proteien 6 (Gas6) en die apolipoproteien E3 (apoE3) is gedemonstreer en gevind om dieselfde area as die laminin interaksie te betrek. Die betrokke interaksie area op AChE het ooreenstemminge met die apolipoproteien interaksie area op die LDL reseptor asook met verwante molekules soos die lae-digtheids lipoproteien reseptor-geassosieerde molekuul (LRP) en die sortilingeassosieerde reseptor (SORL1). Hierdie molekules, insluitend apoE, speel beduidende rolle in die patologie van Alzheimer se siekte. Ooreenkomste tussen AChE en die verwekkings reseptor op myeloïde selle (TREM1) is ook voorgestel, die interaksie is van belang siende dat AChE voorheen geassosieer is met beide haematopoiesis en haematopoietiese kankers. Ko-immuno presipitasie resultate bevestig die AChE-laminin interaksie en dui op die moontlike teenwoordigheid van alternatiewe ligande vir AChE in vivo. In konklusie, karakterisering van die AChE-laminin interaksie areas, gepaard met identifisering van struktureel ooreenstemmende areas in ander molekules, dui op ‘n rol vir AChE in die stabilisering van die basale membraan en verskaf dus ‘n geldige verduideliking vir die oorlewing van die AChE uitklop-muis. Die ooreenstemming van die AChE interaksie area met areas op ander molekules (spesifiek geassosieer met Alzheimer se siekte), asook die moontlike assosiasie van AChE met haematopoietiese differensiering en kanker, lê die grondslag vir verdere ondersoeke.

This thesis introduces a financial simulation model covering two related financial domains: Mobile Payments and Retail Stores systems.   The problem we address in these domains is different types of fraud. We limit ourselves to isolated cases of relatively straightforward fraud. However, in this thesis the ultimate aim is to introduce our approach towards the use of computer simulation for fraud detection and its applications in financial domains. Fraud is an important problem that impact the whole economy. Currently, there is a lack of public research into the detection of fraud. One important reason is the lack of transaction data which is often sensitive. To address this problem we present a mobile money Payment Simulator (PaySim) and Retail Store Simulator (RetSim), which allow us to generate synthetic transactional data that contains both: normal customer behaviour and fraudulent behaviour.    These simulations are Multi Agent-Based Simulations (MABS) and were calibrated using real data from financial transactions. We developed agents that represent the clients and merchants in PaySim and customers and salesmen in RetSim. The normal behaviour was based on behaviour observed in data from the field, and is codified in the agents as rules of transactions and interaction between clients and merchants, or customers and salesmen. Some of these agents were intentionally designed to act fraudulently, based on observed patterns of real fraud. We introduced known signatures of fraud in our model and simulations to test and evaluate our fraud detection methods. The resulting behaviour of the agents generate a synthetic log of all transactions as a result of the simulation. This synthetic data can be used to further advance fraud detection research, without leaking sensitive information about the underlying data or breaking any non-disclose agreements.   Using statistics and social network analysis (SNA) on real data we calibrated the relations between our agents and generate realistic synthetic data sets that were verified against the domain and validated statistically against the original source.   We then used the simulation tools to model common fraud scenarios to ascertain exactly how effective are fraud techniques such as the simplest form of statistical threshold detection, which is perhaps the most common in use. The preliminary results show that threshold detection is effective enough at keeping fraud losses at a set level. This means that there seems to be little economic room for improved fraud detection techniques.   We also implemented other applications for the simulator tools such as the set up of a triage model and the measure of cost of fraud. This showed to be an important help for managers that aim to prioritise the fraud detection and want to know how much they should invest in fraud to keep the loses below a desired limit according to different experimented and expected scenarios of fraud.

Simulation is one approach to analyze and model the real world complex problems. Multi-agent based systems provide a platform to develop simulations based on the concept of agent-oriented programming. In multi-agent systems, the local interaction between agents contributes to the emergence of the global phenomena by getting the result of the simulation runs. In MABS systems, interaction is one common aspect for all agents to perform their tasks. To interact with each other the agents require yellow page services from the platform to search for other agents. As more and more agents perform searches on this yellow page directory, there is a decrease in the performance due to a central bottleneck. In this thesis, we have investigated multiple solutions for this problem. The most promising solution is to integrate distributed shared memory with the directory systems. With our proposed solution, empirical analysis shows a statistically significant increase in performance of the directory service. We expect this result to make a considerable contribution to the state of the art in multiagent platforms.

<p>Liquid chromatography coupling with mass spectrometry (LC/MS) plays an important role in pharmaceutical characterization because of its ability to separate, identify, and quantify individual compounds from the mixture. Polymer brush layer bonded to the silica surface is designed as a novel stationary phase to improve the LC resolution and MS compatibility. The polymer thickness can be controlled to shield the analyte from interacting with the active silanol on the surface and reduce peak tailing. The functional group of the polymer can be changed to tune the selectivity in different separation modes. </p><p> </p><p>Two projects on LC/MS method development for biomolecule characterization using polymer-shell column are discussed in this work. In the first project, a polymer-shell column is used for disulfide bonds and free thiol subspecies identification, which is a major type of structural heterogeneities in IgG1. Compared with commercial columns, the polymer-shell column is able to resolve the free thiol variants without the presence of trifluoroacetic acid and greatly improve the MS signal. In the second project, a polymer-shell column is used for characterizing the drug-loading profile for antibody-drug-conjugates (ADC) via online LC/MS. The separation employs a mobile phase of 50 mM ammonium acetate to keep the ADC intact, and a gradient of water/isopropanol for ADC elution. MS data show that all ADC species remained intact and native on the column. Positional isomers can be separated and identified with the new method as well. Furthermore, to understand the surface chemistry and protein separation behavior quantitatively, a chromatographic simulation study is performed. The result shows that protein separation in RPLC can be described by a bi-Langmuir adsorption isotherm with mixed-mode retention of strong and weak sites. Smaller fractions and lower equilibrium constant of the strong site, which is the active silanol, give less tailing for protein separation.</p>