3^rd International Conference on Genetic and Protein Engineering(10 Plenary Forums - 1 Event) November 08-09, 2017 Las Vegas, USA

Biography:

Marissa L Matsumoto is a Scientist at Genentech, Inc., in the Department of Structural Biology. Her lab focuses on Protein and Antibody Engineering to develop novel research tools for the study of complex ubiquitination events in cell signaling. She has obtained her PhD from Washington University in St. Louis and her BA from Northwestern University.

Abstract:

Statement of the Problem: Ubiquitin is a post-translational modification involved in nearly every signaling pathway. Monoubiquitination occurs when the carboxy-terminus of ubiquitin is linked through an isopeptide bond to a lysine residue on a substrate. Ubiquitin itself contains seven lysine and a free amino-terminus through which additional ubiquitin subunits can be linked, resulting in polyubiquitin chains of different topologies. Determination of polyubiquitin chain linkages requires the use of ubiquitin mutants or complex mass spectrometry experiments. The purpose of this study is to engineer antibodies to detect specific polyubiquitin linkages to provide useful research reagents for the study of ubiquitination in cells.

Methodology & Theoretical Orientation: We used phage display to engineer antibodies with exquisite specificity to the linear, K11, K48, and K63 linkages and used X-ray crystallography to elucidate the nature of their specificity. To detect more complex heterotypic chains containing mixed or branched linkages, we have developed bispecific antibodies using the knobs-into-holes technology.

Findings: The antibodies are highly specific for a given linkage and work in numerous applications, including western blot, immunoprecipitation, and immunofluorescence. Epitope determination by X-ray crystallography demonstrates that rather than contacting the linkage itself, the antibodies recognize a conformational epitope that results from the relative orientation of two ubiquitin subunits because of the spatial positioning of the residue involved in the linkage.

Conclusion & Significance: The polyubiquitin linkage-specific antibodies have provided ubiquitin researchers with an easy-to-use reagent to quickly and efficiently determine the linkages of polyubiquitin chains without the need for ubiquitin mutants or access to mass spectrometry equipment and expertise. These antibodies have become essential tools in studying ubiquitination in cell signaling and have aided in elucidation of numerous pathways including polyubiquitin chain editing, K11-linked chains in cell cycle control, and K11/K48-branched chains in cell cycle and protein quality control.

Biography:

Stephen D Kirby has been working in the field of chemical threats for the US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Grounds for 30 years, to include areas of research such as cyanide detoxification, sulfur mustard metabolism, and edemagenic pulmonary threats. The focus of his current research area has been developing and evaluating catalytic and stoichiometric protein bioscavengers for organophosphorus compounds. Current research has investigated enzyme platforms that include human carboxylesterase, human paraoxonase-1, platelet activating factor acetylhydrolase and the bacterial enzyme phosphotriesterase.

Abstract:

Organophosphorus compounds (OPs) such as sarin and soman are some of the most toxic chemicals synthesized by man. They exert toxic effects by inactivating acetylcholinesterase (AChE) and bind secondary target protein. Enzymes can be engineered by amino acid substitution into OP-hydrolyzing variants (bioscavengers) and used as therapeutics. Some enzymes associated with lipoproteins, such as human plasma platelet-activating factor acetylhydrolase (pPAF-AH), are also inhibited by OPs; these proteins have largely been ignored for engineering purposes, because of complex interfacial kinetics and a lack of structural data. We have expressed active human pPAF-AH in bacteria and previously solved the crystal structure of this enzyme with OP adducts. Using these structures as a guide, we created histidine mutations near the active site serine of pPAF-AH (F322H, W298H, L153H) to generate novel OP-hydrolase activity. Wild-type pPAF-AH, L153H, and F322H have essentially no hydrolytic activity against the nerve agents tested. In contrast, the W298H mutant displayed novel somanase activity with a kcat of 5 min-1 and a KM of 590 μM at pH 7.5. There was no selective preference for hydrolysis of any of the four soman stereoisomers. The kcat/KM for W298H is 8x103 M-1 min-1, a significant enhancement over the wild-type enzyme

Biography:

Karen Bunting is a Science Director at Albumedix, heading Molecular Biology and Fermentation within Research and Technical Development. Prior to the formation of Albumedix, she has joined Novozymes Biopharma UK in 2011 as a Senior Research Scientist and later as Science Manager. She has 20 years’ experience in Structural Biology and Protein Engineering. At Albumedix her primary research focus has been the design and engineering of albumin variants to optimize the circulatory half-life of therapeutic agents. Prior to joining Novozymes, she led a research team at the University of Nottingham focusing on structural analysis of protein-protein and protein-DNA interactions, following on from Post-doctoral work at the Institute of Cancer Research in London. She has obtained BSc in Microbiology from Imperial College, London and PhD in Crystallography from Birkbeck College, London.

Abstract:

Human serum albumin is the most abundant serum protein and has a long history of safe use in biopharmaceutical products. It has many features that contribute to the molecule being naturally well designed as a drug carrier, including its high solubility and stability, long circulatory half-life and the potential to attach therapeutic candidates by fusion or conjugation. We will describe Veltis® albumin variants designed to enhance two of these aspects, long circulatory half-life and drug attachment by conjugation, whilst maintaining solubility, stability and its inherent low immunogenicity. The long circulatory half-life of albumin in the human body (around 19 days) derives in part from the size of the molecule, whereby it resists filtration through the kidneys and in part from its association with the neonatal Fc receptor (FcRn). Albumin binds to FcRn under acidic conditions in the endosome and is rescued from lysosomal degradation back to the plasma where it is released under neutral pH conditions. We will describe our rational design of variants to enhance binding to the receptor under acidic conditions, whilst retaining release at neutral pH, resulting in a greater than two-fold improvement in half-life. Short half-life is a significant challenge for many peptide and protein therapeutics; fusion or conjugation to Veltis engineered albumins offers the potential of monthly dosing. We will also present thio-engineered albumin variants to improve drug efficacy by increasing the drug-albumin ratio whilst maintaining FcRn-binding capabilities and half-life extension. Rational protein design has enabled us to enhance specific properties of albumin related to drug delivery whilst retaining the numerous other features which contribute to the molecule being naturally well designed as a drug carrier.

Biography:

Christopher H Gray has obtained his BSc and PhD from the University of Glasgow, examining the biochemical basis of multidrug resistance in pathogenic fungi. He has then moved to the Institute of Cancer Research in London to gain experience as an X-ray Crystallographer where he was trained in a diverse range of protein production techniques. Following this he took up a position at the CRUK Beatson Institute becoming an early Member of Staff in the Institute’s Drug Discovery Program. As a Structural Biology Team Leader he has day to day responsibility for the protein production, NMR, biophysics and X-ray Crystallography in the program.

Abstract:

The protein production section of the Beatson Institute’s Drug Discovery Program supplies a considerable number of highly purified and active recombinant proteins for structural biology, biophysical and biochemical applications. Pressure to satisfy high demand has prompted the development of novel and innovative methods to streamline workflow maximize output and ensure rapid delivery of critical proteins. Driving up soluble yields, ensuring productive cell culture and reducing the burden on purification resources can enhance the output of a protein production facility. We have substantially improved our productivity by implementing a series of new bacterial MBP fusion vectors. These systems substantially elevate soluble expression but also co-express a protease that removes the MBP tag in vivo, affording the benefits of MBP fusion on soluble yield but eliminating the MBP moiety from downstream purification. As an extension of this, we have developed several systems that allow the monitoring of recombinant expression during fermentation, using an auto-cleaved fluorescent protein tracer. This allows the operator to infer adequate target expression in near real-time, ensuring that cultures allowed occupy fermentation resource lead to satisfactory product yields. We have also accelerated productivity by reducing the number of chromatography steps in common protocols and automating FPLC purification. Taking our inspiration from several emerging and newer multimodal chromatography technologies, we have developed rapid, mixed chemistry affinity strategies to purify tandem tagged proteins. This allows the isolation of high purity target in a single column, often removing the need for slow size exclusion chromatography to polish the product. Finally we have implemented routine multidimensional chromatography on our AKTA AVANT systems performing complex sequences of protein purification and conditioning steps with a minimal requirement for user intervention. The net result is more reliable and frequently higher yield preps, delivered to the downstream user in the shortest possible time.

Biography:

Haiyan has over 19 years of industry experience in the pharmaceutical and diagnostics areas. Haiyan joined Centocor R&D in 2004, which was later named Janssen BioTherapeutics.  In the past 13 years at Janssen Haiyan leads a protein biochemistry group, focusing on developing and optimizing biotherapeutic lead molecules.  She leads the group to work on purification and characterization of monoclonal antibodies, alternative scaffolds including bispecific antibodies, target antigens, receptors and other proteins for various research projects; developing new technologies to improve HTP purification and biochemical QC analyses; aligning with the methods from Discovery to Development process to ensure the smooth transfer of the therapeutic candidates from Lead optimization to Development stages. Haiyan received her Ph.D. in Microbiology at the University of Iowa.  She then took a postdoctoral position at Eli Lilly and Company (Indianapolis).  From 2000 to 2004, Haiyan worked at Roche Diagnostics Division (New Jersey) to lead a group responsible for developing purification process and manufacturing of recombinant DNA polymerases which were used for disease diagnosis.

Abstract:

Monoclonal antibody therapies continue to be the focus of biopharmaceutical industry.  Hundreds of antibodies are in pre-clinical and clinical development. Purification and characterization of the lead molecules is vital during the lead optimization and candidate selection stage.  Protein A affinity chromatography and additional polishing steps are used to purify panels of antibody hits against specific target.  Throughout the process, biochemical and biophysical assessment for developability and manufacturability are applied to select lead candidate.  This talk will review the methods, the challenges, and how the purification and characterization data contributes to lead selection.  A case study will be discussed on a small scale purification method scouting for difficult proteins which facilitated the development of the large scale process and shortened development timeline.

Biography:

Jeff Mihailoff has an MS in Molecular Biology and MBA from the University of Florida. Since leaving academia, he has been selling research tools into the Biopharma and Academic research market. Prior to ForteBio, he was the bioprocess and chromatography specialist at GE Healthcare focused on downstream purification. He also has experience with multiple different bioanalytical techniques such as Surface Plasmon Resonance, Isothermal Titration Calorimetry, and HPLC. He and his wife enjoy scientific advancement and have dedicated their lives to assisting scientists in taking their technology to the next level.

Abstract:

Pall Fortebio’s Bio-Layer Interferometry has been utilized by the antibody biopharma market since its inception in 2005. This game-changing technology allowed scientists to bypass ELISA and HPLC technologies for antibody quantification. In addition, its lower price point and easy of use allowed it to be a substitute for Surface Plasmon Resonance (SPR) studies on antibody-antigen binding affinity. However, there have remained questions about the accuracy of the work as related to SPR. In this talk, the author will review a recent large-scale study in which different variables related to assay design were tested and then compared to SPR studies. Finally, the author will review some of the newer technology changes and how they are being applied to bioprocess residuals quantification.

Biography:

Thomas Rohrer is Associate Director of Bioconjugate Commercial Development at Lonza and has over 30 years of experience in biotherapeutic process development, scale-up and manufacturing. He holds a BS in Biochemistry and MS in Chemical Engineering. He established process development and clinical manufacturing of ADC(s) at Cambrex Biopharma in 2005. After Cambrex Biopharma was acquired by Lonza in 2007 he joined the Lonza Exclusive Synthesis ADC business team in Visp, Switzerland. Prior to joining Lonza he held senior positions in biotherapeutics process development and manufacturing at Human Genome Sciences, Otsuka Pharmaceutical and the National Cancer Institute (NCI) - Frederick Cancer Research Facility. 

Abstract:

Advances in the coupling of antibodies to potent cytotoxic drugs have resulted in stable delivery platforms with improved pharmacokinetics, which spares patients from the debilitating systemic toxicity observed with traditional chemotherapy. Two ADCs Adcetris® (brentuximab vedotin) and Kadcyla (trastuzumab emtansine) are currently approved for difficult -to-treat therapeutic indications. Currently three more ADCs, Inotuzumab ozogamicin (Pfizer),Vadastuximab talirane (Seattle Genetics) and Depatuxizumab mafodotin (AbbVie) are undergoing regulatory review or completing Phase III clinical studies. In all, more than 60 ADCs are in clinical trails with over 100 more in the pipeline. In addition to ADCs the multi-faceted field of bioconjugates also extends to coupling targeting agents to non-cytotoxic payloads. Perserving the targeting ability of antibody and the effector function of the payloads require that process R&D find the intersection of reaction parameters which preserve the integrity of both molities. Case studies will be presented which discuss the development and manufacturing of several next generation bioconjugates. 

Biography:

Victor Guzov has his expertise in protein engineering and evolution, mode of action of insecticidal proteins and genetic engineering of crop plants. He has been long involved in optimization of insecticidal proteins for the purpose of protection of crop plants from insect infestation using biotechnology approaches. That led him to research on mode of action of insecticidal proteins, and ways to assess their value for insect resistance management. His collaborative research with both industry and academic scientists on evolution of Bt proteins resulted in new opportunities for effective management of insect resistance to genetically-modified crop plants.

Abstract:

The development of insect resistance to insecticidal proteins, such as Bt proteins, is a continuous threat to insect control traits in biotech agricultural crops. This requires constant search for insecticidal proteins with new modes of action (MOA), which enhance insect resistance management options via delaying new resistance development and combating already existing resistant insects. This presentation will describe the successful onboarding of phage-assisted continuous evolution (PACE) technology, and its application to Bt toxins for generating new MOAs.

Biography:

Yvette Stallwood has completed her PhD at the University of Birmingham (UK) and joined Lonza in 2007. She has been Head of Applied Protein Services for five years. The Applied Protein Services team are focussed on the development and provision of services to support the development of new biotherapeutic proteins with a particular focus on immunogenicity and manufacturability.

Abstract:

This presentation will discuss how in silico and in vitro methodologies are employed to perform a developability and immunogenicity risk assessment in order to highlight potential risks of failure for the development of biotherapeutics. In silico methods can be used to evaluate protein sequence and structure to assess the likelihood of immunogenic responses and potential manufacturability issues including aggregation and PTMs. Ex vivo T and B-Cell responses enable the assessment of overall immunogenicity risks; different approaches are highlighted to further identify processed and presented epitopes.

Biography:

Franz Grus is the Head of Experimental Ophthalmology, Department of Ophthalmology in Johannes Gutenberg University Medical Center, Germany. His area of interest includes: Glaucoma, age-related macular degeneration and dry eye biomarker research, high-throughput-research methods, proteomics and immunology, diagnostic technologies, antibodies in diagnosis and treatment in personalized medicine. He has been in positions like: Principal investigator for several research projects funded by the German Research Foundation (DFG) and the German Society of Ophthalmology (DOG), as well as other institutions and companies.

Abstract:

In glaucoma, the elevated intraocular pressure cannot explain the disease in all patients. Glaucoma is a neurodegenerative disease, leading to the loss of retinal ganglion cells (RGC). Immunoproteomics could play a significant role. Several studies could provide hints for an involvement of autoantibodies in the pathogenesis of the disease. One of those candidates is several heat-shock proteins AAB. The complex profiles of antibodies were analyzed by mass spectrometry based techniques and customized antigen microarrays of 40 different antigens in more than 1000 patients. The resulting profiles were analyzed by different data mining techniques such as artificial neural networks. In all studies we could demonstrate consistent up- and down-regulations of immune reactivities against ocular antigens in glaucoma. The glaucoma could be recognized by a sensitivity and specificity of more than 90%. Furthermore, these antibodies could be useful as an innovative glaucoma treatment option. We analyzed in a glaucoma animal model the effect of an intravitreal injection of anti-synuclein antibodies. Axon density/mm² showed a decay (p<0.01) in controls, (p<0.01) in buffer group, and (p=0.19) in the α-synuclein group. Mass spectrometry revealed changed levels of CCDP93 (-2.6x), cofilin-1 and reticulon 4 (both -2.5x) in IOP elevated eyes and peripherin-2 (2.4x), cofilin-1 (3.5x) and malate dehydrogenase (11.9x) in α-synuclein treated eyes. The results of this study demonstrate clearly that antibody patterns could be useful for diagnosis especially if transferred to a point-of-care device such as lateral flow assays, but also given intravitreal, a promising new approach for neuroregenerative treatment in personalized medicine in glaucoma.

Biography:

Anna V Hine has studied at the University of Manchester (UK) and Harvard Medical School. She is a Reader and Associate Dean Enterprise at Aston University (UK). In March 2013, she was named BBSRC Commercial Innovator of the Year 2013, for her work in transferring ‘ProxiMAX randomisation’ into SME Isogenica Ltd. She is a Molecular Biologist by training.

Abstract:

ProxiMAX and MAX randomization technologies are defined saturation mutagenesis processes that deliver precision control of both identity and relative ratio of amino acids at specified locations within a protein library. Both processes are non-degenerate, meaning that encoding DNA libraries are as small as is physically possible. ProxiMAX is the technology that lies behind Isogenica’s Colibra™ offering and is ideal for saturating contiguous codons, as required in antibody libraries. In contrast, ‘MAX’ randomization targets codons at disparate locations within a gene and is therefore more applicable to other scaffolds or proteins. Since no constraints are imposed by the genetic code, both technologies can eliminate unwanted amino acids such as cysteine and methionine from libraries or encode desired subsets of amino acids with ease. Yet their underlying processes are quite different. This presentation will examine the development of both ProxiMAX and MAX randomization process and give examples of libraries created to date.

Biography:

Stefan Zielonka has received his PhD from the Technische Universitaet of Darmstadt, Germany, where he worked in the Group of Harald Kolmar in the field of Protein Engineering of non-canonical antibodies. Now, he works as Senior Scientist at Merck KGaA (EMD Serono), Germany, in the Department Protein Engineering and Antibody Technologies (PEAT). He was Fellow of the Merck’sche Gesellschaft fuer Kunst und Wissenschaft e.V. and was awarded with the Kurt-Ruths-Award (2016) and the Rainer-Rudolph-Award in Biotechnology and Protein Chemistry (2016).

Abstract:

Yeast surface display has proven to be a versatile platform technology for antibody engineering enabling online and real-time analysis as well as characterization of library candidates. During this talk, the process of antibody library generation will be explained as well as selection of target-specific antibodies with prescribed properties since desirable features such as species cross-reactivity can be implemented into the screening procedure using FACS. Additionally, a novel streamlined one-step approach for the generation of yeast surface display Fab libraries will be discussed that allows for simultaneous introduction of heavy chain and light chain variable regions into one single display vector. Finally, a generic approach for the generation of human IgG-like bispecific antibodies will be presented that relies on the combination of immunization of transgenic rats with yeast surface display.

Biography:

Lia Monica Junie is the Head of the Department, also coordinating the activities of both Laboratory Medicine specialty resident doctors and PhD doctor’s thesis in the medicine field. She is a Member in the Board of Scientific Societies, Reviewer in many peer-reviewed journals, Expert of the Ministry of Education and Research and Evaluator. She has coordinated research projects, published books and more than 200 scientific articles in prestigious journals. She has organized and attended  numerous national, international congresses, as President, Member in the Organizing Committees, Invited speaker, Keynote speaker or Chairperson

Abstract:

Background: Staphylococcus aureus infections remain a serious medical problem and prompt initiation of appropriate antimicrobial chemotherapy is important to improve the prognosis of these infections.

Methods: The strains were isolated from hospitalized patients in Universities Hospitals, Cluj Napoca, Romania, during 2016. Species identification was achieved by standard procedures and by the automated system Vitek 2 (Biomérieux). The susceptibility to antibiotics was performed using the disk diffusion method (Kirby-Bauer) according to the CLSI criteria and by the Vitek 2 System, for a total of 855 strains.

Results: From all isolated strains, 51 (5.9%) yielded Staphylococcus aureus. 34.3% of the S. aureus isolated strains were MRSA. 47.1% of the S. aureus tested strains were resistant to clindamycin. Moderately increased percentages of resistance were found by testing the strains to ciprofloxacin (20.3%). 16.7% of all the strains were resistant to gentamicin. Low levels of resistance were observed by testing the strains to trimethoprim/sulfamethoxazole (3.9%). All the S. aureus strains were sensitive to glycopeptides and the second line anti-Staphylococcal antibiotics: linezolid, quinupristin/dalfopristin.

Conclusion: The majorities of the isolated strains were MSSA and exhibited higher percentage susceptibility, in contrast to those that had been isolated in 2015. Therefore, the policy that was adapted to avoid the spread of resistant strains was effective and recommended the need to introduce the antibioticotherapy control program in the Romanian hospitals.

Biography:

D L Savithramma has completed her PhD from University of Agricultural Sciences, Bangalore, India and Post-doctoral studies as a Biotechnology National Associate from Indian Institute of Science, Bangalore, India. She is a Professor and University Head of Genetics and Plant Breeding at University of Agricultural Sciences, Bangalore, India. She has guided 13 PhD students and 30 Master’s students in Genetics and Plant Breeding. She has published more than 70 papers in reputed journals and has released eight varieties in vegetable cowpea, seed cowpea, peanut and Chrysanthemum.

Abstract:

Tomato is an important commercial vegetable crop. However, tomato is affected by both biotic and abiotic stresses, most importantly; the increasing occurrence of drought worldwide has highlighted the need for development of drought tolerant genotypes in tomato. Identification and selection of genotypes with improved drought tolerance will play an important role in developing tomato genotypes with better yield. The aim of this study was to investigate the effects of drought stress on fruit yield through physiological traits related to drought tolerance viz., SPAD chlorophyll Meter Reading (SCMR), Specific Leaf Area (SLA), Relative water content (RWC) and leaf rolling (LR) and also establish marker association with physiological and fruit yield traits. One hundred (100) germplasm accessions of six cultivated tomato and related species along with three check varieties were evaluated during summer 2014 and 2015 under well watered and water stress condition (stress was imposed on 60^th day after transplanting for a period of 20 days). Population structure was determined using model based method by structure, Neighbor-joining method, Q-matrix population structure and Delta K analysis and all of them separated population into four clusters. Marker-trait associations are established using 145 published SSR markers with syntenic Linkage Disequilibrium (LD) values (r2=0.09) which depicted that some markers detected as most powerful due to high R2 value. Marker SSR 52 associated with SCMR and TGS 2002 for fruit yield per plant and clusters per plant in lycopersicum species. The markers SSR 14 and LELEUZIP for LR, LEet 004 for plant height, fruit yield per plant and LR in cherry species. The marker SSR 593 for days to 50% flowering, SSR 27 for days to first fruit set, SSR 218 for stem girth, TGS 0412 for clusters per plant, SSR 128 for fruits per plant, SSR 599 and SSR 593 for fruit yield in all the spp. This study demonstrated that association mapping in cultivated and related species can enhance the information from QTL studies towards the implementation of marker-assisted selection. From the present study five germplasm accessions namely LA 2976, WIR 13708, EC 676809, EC 677123 and EC 771596 were identified as top drought tolerant genotypes which may be used as parents in hybridization program to develop drought tolerant and high yielding varieties in tomato.