BioMAN Summit - November 14, 2014

November 14, 2014

1.	Inhibitor mediated Bcr-Abl degradation: A novel therapeutic approach for Chronic Myeloid Leukemia
	Arifa Sayyidah Ahsan¹, Marcus J. C. Long², and Lizbeth Hedstrom³
	¹Undergraduate Program in Biochemistry, Brandeis University, Waltham 0245, USA ²Graduate Program in Biochemistry, Brandeis University, Waltham 0245, USA ³Departments of Biology and Chemistry, Brandeis University, Waltham 0245, USA
	Bcr-Abl, a fusion oncogenic protein with deregulated tyrosine kinase activity, is the pathophysiological cause of chronic myeloid leukemia (CML). Current treatment of CML with tyrosine kinase inhibitor drugs like Imatinib (Gleevec, Novartis) is not always effective since some of the downstream signaling pathways of Bcr-Abl are kinase-independent. This problem can potentially be resolved if elimination, instead of inhibition, of Bcr-Abl could be achieved. The Hedstrom Lab recently discovered that proteins tagged with the hydrophobic molecule, Triboc-Arginine (Boc3Arg), are degraded by the proteasome. In order to evaluate the efficacy of Boc3Arg tag as a degron of Bcr-Abl, we designed a new inhibitor, AS-22, by linking Boc3Arg to GNF-2, which belongs to a new class of allosteric inhibitors of Bcr-Abl. A protein degradation assay was performed with the lysate of BaF3.p210 cells which express Bcr-Abl. Western blot analysis showed that a 60 min incubation of BaF3.p210 lysate with GNF-2 brought about a 25±13 % (N=2) reduction in the amount of Bcr-Abl present whereas the percentage of reduction shown by AS-22 was 73±11% (N=5). The results thus look promising and this project may not only contribute towards finding a better drug for CML, but also serve as an alternate model for cancer therapy.
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2.	In-process Virus Tests as Forward Processing Decision Tool Enhance Containment of Virus Contaminations in Cell Culture Based Biomanufacturing
	Paul W. Barone^1, James C. Leung^1, Stacy Springs^1, and Michael E. Wiebe^1
	¹Center for Biomedical Innovation, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA ^*Equal contribution first authors
	Virus contaminations in the manufacturing of recombinant therapeutics and vaccines are rare events but may have a severe impact on operations. One of the factors that modulates the severity of the impact is the ability to contain the contamination. This can help to restrict the scope and magnitude of clean-up, restart and the investigation. More importantly, successful containment prevents the adventitious agent from reaching the final drug product. The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) gathered data from biopharmaceutical manufacturers to verify that incorporation of virus tests as "forward processing" criteria enabled containment of virus contaminations to the upstream process of cell culture based Biomanufacturing. Many manufacturers are implementing rapid virus detection methods, such as PCR, to gain real-time level response for process forwarding decisions.
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3.	A Pharmacokinetic Model of a Tissue Implantable Insulin Sensor
	Gili Bisker¹, Nicole M. Iverson², Jiyoung Ahn¹, and Michael S. Strano¹
	¹Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA ²Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
	While implantable sensors such as the continuous glucose monitoring system have been widely studied, both experimentally and mathematically, relatively little attention has been applied to the potential of insulin sensors. Such sensors could provide feedback control for insulin infusion systems and pumps, and provide platforms for the monitoring of other biomarkers in vivo. In this work, we develop the first pharmacokinetic model of an affinity sensor for insulin operating subcutaneously in the limit of where mass transfer across biological membranes reaches a steady state. Using a physiological, compartmental model for glucose, insulin, and glucagon metabolism, the maximum sensor response and its delay time relative to plasma insulin concentration, are calculated based on sensor geometry, placement, and insulin binding parameters for a sensor localized within adipose tissue. A design relation is derived linking sensor dynamics to insulin time lag and placement within human tissue. The model should find utility in understanding dynamic insulin responses and forms the basis of model predictive control algorithms that incorporate sensor dynamics.
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4.	Rational design of peptides for affinity purification of biologics
	Divya Chandra^1, Steve Timmick^1, Chaz Goodwine¹, Divya Shastry², Steven Cramer¹, and Pankaj Karande¹
	¹Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy NY 12180 ²Department of Biochemistry and Biophysics, Rensselaer Polytechnic Institute, Troy NY 12180 ^*Equal contribution first authors
	Current strategies for downstream processing of biologics involve lengthy purification trains making the entire process very time and resource-intensive. Affinity chromatography, on the other hand, provides several advantages over traditional chromatography procedures by limiting the number of steps involved. However, affinity chromatography has found limited commercial use due to the lack of robust, selective, cost-effective, and high affinity ligands for the target biologic. In this work, we will demonstrate the design of affinity peptides for the purification of human growth hormone (hGH) produced in Pichia pastoris. Peptides as affinity ligands offer several advantages such as amenability to rational design and modification, ease of manufacture, low cost, high stability and prolonged shelf-life. Using high-throughput screening in concert with a diverse array of peptide design strategies, we have designed libraries of peptides and identified candidates with orthogonal properties under a given set of operating conditions. The designed peptide libraries exhibit a range of affinities, selectivities and elution characteristics for the target biologic, thus enabling the selection of peptide ligands that can be used for affinity-based purification of hGH. Overall, this work demonstrates the potential impact and power of rational design of affinity ligands on commercial bio-manufacturing.
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5.	Size-Based Biomolecule Preconcentration and Separation Using Slanted Nanofilter Array
	Sung Hee Ko¹ and Jongyoon Han^1,2,3
	¹Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA ²Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA ³BioSystems and Micromechanics (BioSyM) IRG, Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore
	Size-based separation of proteins (e.g. SDS-PAGE) is widely used to check the purity of protein drugs in pharmaceutical manufacturing process. While liquid gel electrophoresis has been automated in a microfluidic platform, this technique still requires polymeric sieving matrices that can increase technical complexity, preventing implementation of truly portable, on-site drug purity and efficacy tests. We have designed a novel nanofilter-based, continuous-flow preconcentration and separation chip. Our design relies on angled nanofilters and enables both protein preconcentration and separation with a simple one-step introduction of analytes. Proteins and other biomolecules are first manipulated to form a narrow, concentrated band, followed by size-based separation for analysis. Biomolecule preconcentration and separation was achieved successfully with better separation efficiency and lower detection sensitivity than previously developed nanofilter sieving system. We believe the device demonstrated here can be used for on-site biomolecule analysis, such as biologics (protein drugs) purity and efficacy monitoring system, due to its operational simplicity, robustness (no degradation of gels) and minimal sample use (~1nL of sample volume). The high detection sensitivity of the system could enable detection of low-level impurities in biologics drug that can lead to significant toxicity in patients.
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6.	UPLC-MS/MS analysis of glycoprotein changes in Mouse kidney cells with the phenotype of autosomal dominant polycystic kidney disease
	Yan-Jun Liu¹, Anna Fan Zhang¹, Shiaw-Lin Wu¹, Yunjoon Jung², Shan Qin², William S. Hancock¹, and Jordan A. Kreidberg^2,3
	¹Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115, USA ²Department of Medicine, Boston Children's Hospital, and Department of Pediatrics, Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts 02115, USA ³Harvard Stem Cell Institute, 1350 Massachusetts Avenue, Cambridge, Massachusetts 02138, USA
	Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most prevalent, potentially fatal disorders. In previous study, some biomolecular changes have been observed in this disease, including glycosylation. Glycoproteins participate in many key biological processes such as cell adhesion, receptor activation, and signal transduction. Determination of ADPKD related glycoprotein differences can unveil significant information for the study of disease mechanisms, which is essential to improve diagnosis and treatment strategies. In this study, ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) was used to recognize ADPKD related changes in glycoproteins. Lectin Sambucus nigra bark (SNA) was used to enrich glycoproteins which have the changes of sialic acid content. Subsequently, 1D SDS PAGE was performed followed by in-gel digestion and UPLC-MS/MS analysis. As an example of our results, clinically interesting glycoproteins, such as Thrombospondin-1 (TSP-1) and cell surface glycoprotein MUC18, were only detected in the disease sample and will be targeted for future study of glycan motifs associated with ADPKD.
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7.	Tunable Staged Release of Therapeutics from Layer-By-Layer Coating with Clay Interlayer Barrier
	Jouha Min^1,2, Richard D. Braatz¹, and Paula T. Hammond^1,2
	¹Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA ²David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
	In developing new generations of coatings for medical devices and tissue engineering scaffolds, there is a need for thin coatings that provide controlled release of multiple therapeutics. For orthopedic applications, a promising strategy to accelerate implant-tissue integration and improve surgical outcomes is to deliver an antibiotic and a growth factor for healing bone defects while preventing infection at the implant site. To this end, a series of self-assembled, polymer-based conformal coatings, built by using a water-based layer-by-layer (LbL) assembly techniqueÑa method involving the alternate adsorption of oppositely charged polymers—was generated as biomimetic implant surface coatings that are conformal and only microns in thickness. These LbL coatings consist of two parts: a base osteoinductive component containing bone morphogenetic protein-2 (rhBMP-2) beneath an antibacterial component containing gentamicin (GS). For the fabrication of compartmentalized composite films with controlled and staged release profiles, we presented a new strategy—implementation of laponite clay barriersÑthat allows for a physical separation of multiple components by controlling interlayer diffusion and thus enabling separation of the components between different therapeutic containing multilayers. To our knowledge, this is the first study demonstrating that multi-drug LbL coatings can be tuned for specific orthopedic applications involving infection treatment and bone regeneration. The general demonstration of controlled multicomponent release from a thin film coating platform is a significant advance for a variety of surface delivery applications.
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8.	Protein quality testing by a microfluidic electrokinetic preconcentrator
	Wei Ouyang¹, Sunghee Ko¹, and Jongyoon Han^1,2
	¹Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA ²Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
	Protein quality, including activity, purity and sterility, is of essential importance in drug manufacturing. This work develops a microfluidic electrokinetic platform for rapid testing of protein quality. In our device, a nanojunction was embedded in a microfluidic channel to realize preconcentration of charged molecules (e.g. protein) utilizing the ion concentration polarization phenomenon. Protein variants, including oxidized, deamidated, denatured protein and protein bound to sensing molecules, demonstrated different electrical mobility with the original protein. Based on the different preconcentration positions due to different electrical mobility, the activity and purity of protein was detected and quantified. Human Growth Hormones (hGHs) by Novo Nordisk and Raybiotech were tested as sample drugs. (1) Purity. The purity ratio of Novo hGH was tested to be 89.2%, while Raybiotech was 40.3%. (2) Denaturation. Fresh Raybiotech hGH had a purity ratio of 40.3%, while heat denatured Raybiotech hGH had a purity ratio of 18.5%. (3) Activity. Based on a primary result of hGH receptor assay in our device, Novo hGH demonstrated good functioning activity.
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9.	Chemistry, Analysis and Biology of Protein Modifications: Methylation and Isoaspartic Acid
	Wanlu Qu¹, Min Liu¹, Bobby Lee¹, and Zhaohui Sunny Zhou¹
	¹Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA
	Elucidation of the biochemical activity and specificity of enzymes is an essential step towards understanding their physiological roles. Despite the progress in genomics, structural proteomics, and computational biology, functional annotation of enzymes is often limited to the family level. This challenge is exemplified by the large and diverse family of S-adenosyl-methionine dependent methyltransferases (AdoMet or SAM-MTases).The precise substrate specificity for MTases remains largely undefined due to the intrinsic chemical properties of the methyl group: it is small (15 Da), free of charge and inert. Based on the concept of multi-substrate adduct inhibitors, we have devised a new AdoMet analogue that contain electrophilic "hook"to "catch" the nucleophilic substrates. More importantly, the new AdoMet analogue is an efficient tool to identify substrates of methyltransferases, even unknowns. Isoaspartic acid (isoAsp or isoD) formation is another ubiquitous yet underappreciated post-translation modification, which is generated via the deamidation of asparagine or isomerization of aspartic acid in proteins. An unbiased screening of isoAsp at low abundance remains challenging with the smallest PTM by mass changes. To this end, chemo-enzymatic tagging and affinity enrichment as well as sample enrichment via selected digestion followed by identification using electron-transfer dissociation (ETD) mass spectrometry were reported.
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10.	Real time identification of therapeutics using Raman spectroscopy
	Gajendra P. Singh¹, Ningren Han¹, and Rajeev J. Ram¹
	¹Physical Optics and Electronics Group, Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
	Real time identification of biologics currently requires expensive biochemical analyses and instruments. Moreover, they are time consuming and laborious. Raman spectroscopy, which makes use of inelastically scattered light and can provide a biomolecular fingerprint, is one of the most promising optical techniques in this regard. We show how confocal Raman spectroscopy can identify in real time two different biologics in only a few microlitres of sampling volume. We also show that the concentration of human growth hormone of as low as 75 micrograms per millilitre can be detected using our system. We are currently designing and building a Raman flow cell that can be integrated online during the manufacturing process.
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11.	Spectroscopic approach for dynamic bioanalyte tracking with minimal concentration information
	Nicolas Spegazzini^1*, Ishan Barman², and Ramachandra R. Dasari¹
	¹Laser Biomedical Research Center, Massachusetts Institute of Technology, Cambridge, MA 02139, USA ²Department of Mechanical Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA ^*Correspondence: Nicolas Spegazzini, e-mail: nspega@mit.edu
	Vibrational spectroscopy has emerged as a promising tool for non-invasive, multiplexed measurement of bioanalyte - an outstanding problem in biophotonics. Here, we propose a novel analytical framework that enables spectroscopy-based longitudinal tracking of chemical concentration without necessitating extensive a priori concentration information. The principal idea is to employ a concentration space transformation acquired from the spectral information, where these estimates are used together with the concentration profiles generated from the system kinetic model. We employ this new method in two different areas 1) real time release pharmaceutical applications and 2) biomedical area. 1) Biological drugs are most often produced by cells growing in a bioreactor, a vat designed to maintain carefully calibrated conditions. Because the cells are alive, every time you run a reactor, the result can be a bit different. Using Raman spectroscopy to the simultaneous quantitative determination of bioanalyte in batch process was demonstrated in situ in bioreactors. 2) Using blood glucose monitoring by Raman spectroscopy as an illustrative example, we demonstrate the efficacy of the proposed approach as compared to conventional calibration methods. This method offers a new route at screening gestational diabetes and opens doors for continuous process monitoring without sample perturbation at intermediate time points.
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12.	Komagataella Phaffii Pastoris Host Cell Protein Characterization
	KyOnese Taylor¹, Jared Auclair¹, and William Hancock¹
	¹Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, USA
	MIT is leading a team of researchers to develop the Integrated and Scalable Cyto-Technology (InSCyT) platform. The platform was developed for the production of on-site protein therapeutics. Before the initial product genes of hGH and IFN_2b are introduced, the Saccharomyces Cerevisiae host organism strain, Komagataella phaffii pastoris(k.phaffii), must be deciphered entailing all of its Host Cell Proteins(HCPs) that could contaminate the desired protein therapeutic. The goal of my project, is to characterize the secreted fraction of the k.phaffii Null strain NRRL-11430. Within the Barnett Institute, we use the bioanalytical application of SDS-PAGE, in-solution digestion (InSD), RP-HPLC-MS/MS(q-tof), to monitor the HCPs. To increase the dynamic range of the characterization study, we identified HCPs in both unfractionated Null and eight anion exchange(AEX) fractions. These proteins are being identified by different bioinformatics database searches within Proteinscapeª. Once the lists of HCPs are obtained, we will then have the secreted proteome for k.phaffii, which can be compared with the secretome of product producing strains. The resulting data once deposited in online databases, will serve as a resource for biotechnology applications of this yeast production system.
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13.	Integrated and Scalable Cyto-Technology (InSCyT) Platform for Monitoring two-chain Form of Biopharmaceutical Manufactured Recombinant Human Growth Hormone using LC-MS
	Annie Yu Wang¹, Shiaw-Lin Wu¹, and William S. Hancock¹
	¹Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, USA
	Cleavage of protein therapeutics by internal proteinase in host produced using recombinant DNA technology is inevitable yet underappreciated. It may cause biomanufacture inconsistency and affect the stability and activity of the product. Two-chain recombinant human growth hormone (rhGH) is a proteolytically form of rhGH and was found back in 1990s. Determination of cleavage site is of great significance as it can provide information of the enzyme type causing the internal cleavage and allow further optimization of the biomanufacture process. Although it is reported that the cleavage site of two-chain rhGH locates mostly in the surface loop in the 3D structure of rhGH, few reports confirm the cleavage site conclusively using advanced techniques. Liquid chromatography coupled online with mass spectrometry is a state of art tool to characterize proteins with high sensitivity and accuracy. In this study, we employ bottom up strategy using multi-enzyme digestion approach to characterize the two-chain rhGH using LC-MS. Data base search and manual inspection are used to confirm the cleavage site.
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14.	Harnessing Amino Acids Mass Balance for Screening Mammalian Cell Lines with Recombinant Proteins as Products
	Wen Wang¹ and Daniel I. C. Wang¹
	¹Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
	Post-transfection clone screening is a crucial step in mammalian cell line development for production of therapeutic proteins. Ideally, the screening should be non-invasive, economical, and effective. In this poster, we report a fundamentally new method for predicting mAb titer in a Chinese hamster ovary (CHO) cell culture system based on mass balance of two essential amino acids. For proof of concept, 24 clones were tracked and characterized. The ranking results of this new method were compared with the ELISA ranking results, which showed that our method could accurately predict the top 20% producers in a heterogeneous CHO clone pool. This study provides an alternative way to screen high therapeutic protein producers in a low-cost manner. This screening method should also be applicable to other mammalian cell lines for production of versatile therapeutic proteins.
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15.	Analysis of Intact Recombinant Human Growth Hormone (rhGH) by Collision Induced Dissociation (CID) FTMS
	Di Wu¹, Yang Tang¹, Shaw-Lin Wu¹, Jeff Agar¹, and William Hancock¹
	¹Barnett Institute and Dept. of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02215
	Mass spectrometry (MS) has been widely util ized in biological and biomedical area. The identification and characterization of proteins are necessary in modern biopharmaceutical studies. The majority of experiments use the "bottom-up" mass spectrometry methods, however, top-down analysis, both its methodology and technology, are developed continuously, leading to increased usage of this strategy. This study compares top-down with bottom-up analysis on recombinant human growth hormone by CID-FTMS. Utilizing computational tools, including the database program Mascot. In this study, results from top-down MS were confidently assigned and show that top-down and bottom-up provide complementary sequence coverage. This study has involved an orthogonal workflow coupling with RP-HPLC with a C4 column. Although top-down mass spectrometry assay still has further room for development, this method demonstrated its sensitivity, accuracy and efficiency. The new technique will allow the determination of difficult to determine variants such as N-terminal and 2-chain species in an efficient way in the future.
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16.	Cooperative Co-culture of E. coli and S. cerevisiae for Overproduction of Paclitaxel Precursors
	Kang Zhou¹, Kangjian Qiao¹, Steven Edgar¹, Gregory Stephanopoulos¹
	¹Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
	Metabolic engineering of microorganisms such as Escherichia coli and Saccharomyces cerevisiae to produce high-value natural metabolites is often done through functional reconstitution of long metabolic pathways. Problems arise when parts of pathways require specialized environments or compartments for optimal function. Here we solve this problem through co-culture of multiple engineered organisms, each of which contains the part of the pathway that it is best suited to hosting. In one example, we divided the synthetic pathway for paclitaxel precursors into two modules, expressed in either S. cerevisiae or E. coli, and cultured the microorganisms in the same bioreactor. Stable co-culture was achieved by designing a mutualistic relationship between the two species in which a metabolic intermediate produced by E. coli was used and functionalized by yeast. This synthetic consortium efficiently produced oxygenated taxanes, tanshinone precursors and functionalized sesquiterpenes.
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