1
SUNDAY MORNING
8:20 – 10:40 am BCEC 108
Stem Cells: Quantifying, Modeling and Controlling Stem Cell Fate J. M. Piret, Organizer Papers 1-7
BIOT 1 – Behavior-activated cell selection: Application to stem cell expansion
Brad Dykstra1, John Ramunas2, David Kent1, Connie J. Eaves1, and Eric Jervis2. (1) Terry Fox Laboratory, British Columbia Cancer Agency, 675 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada, (2) Department of Chemical Engineering, University of Waterloo, 200 University Ave. West, Waterloo, ON N2L 3G1, Canada,
The nondestructive nature of cellular behavior analysis using long-term imaging facilitates cell classification. To look for new indicators of self-renewing capacity, hematopoietic stem cells (HSCs) isolated from adult mouse marrow were individually micromanipulated into the wells of a microarray imaging chamber and then cultured for 4 days with each cell and its progeny being imaged using time-lapse photography. Individual clones were then harvested and assayed in mice for HSC compartment repopulating activity. Characteristics identified from image sequences were screened and 3 were found to be predictive of HSC potential: mitotic activity, longer cell cycle times, and uropodia. Clone selection criteria developed using these data allowed all HSC-containing clones to be detected and at a 2.4-fold greater efficiency in subsequent experiments. The BACS platform represents an advance towards the goal of dynamic single cell analysis in heterogeneous populations and demonstrates the potential of this technology for preparative cell isolation devices.
BIOT 2 - Tracking stem cell fate using a cell-based microscale platform
Tiago G Fernandes1, Seok Joon Kwon1, Moo-Yeal Lee1, Margarida M Diogo2, Cláudia Lobato da Silva2, Joaquim MS Cabral2, and Jonathan S. Dordick1. (1) Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Center for Biotechnology and Interdisciplinary Studies, 110 8th Street, Troy, NY 12180-3590, Fax: 518-276-2207, , (2) IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Lisboa 1049-001, Portugal
Stem cells are potential sources for clinical applications. However, their therapeutic use is contingent upon precise control over the cell population during their in vitro expansion and differentiation. We have developed a miniaturized 3D cell-culture based chip for high-throughput screening. The cell chip consists of mouse embryonic stem (mES) cells encapsulated in 20nL alginate gels arrayed on a functionalized glass slide. Our results show that this platform is suitable for studying the expansion of mES cells, while retaining their pluripotent and undifferentiated state. Moreover, it provides a suitable system for the high-throughput screening of small molecules and their combinations that direct the fate of stem cells in a spatially addressable manner. This screening was aided by the development of a microarray in-cell Western technique that provides highly quantitative information on cell function. We expect this work to impact the design and control of stem cells for tissue engineering and biological studies.
BIOT 3 -Exploiting the synergy between lysate and acoustic standing wave fields to increase the retroviral transduction of suspension cells
Pascal R. Beauchesne1, Venkata S. Tayi1, Bruce D. Bowen2, and James M. Piret1. (1) Michael Smith Laboratories & Department of Chemical and Biological Engineering, University of British Columbia, 2185 East Mall, Vancouver, BC V6T 1Z4, Canada, Fax: 604-822-2114, , (2) Department of Chemical and Biological Engineering, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
Recombinant retroviruses are effective therapeutic transgene delivery vehicles for stem cell-based gene therapy. However, the short half-life (~6 h) and low diffusivity (~6x10-8 cm2/s) of retroviruses often contribute to limited transduction efficiency. We have explored using whole cell lysate to improve the transduction efficiency of K-562 and TF-1 cells in MSCV-IRES-GFP virus-containing medium. Lysate addition increased transduction efficiency by 6-fold in t-flasks as well as in stirred suspension spinners and was a function of both lysate and target cell concentrations. When used in combination with a 1.95 MHz acoustic resonance filter, in which cells are retained within the pressure node planes while lysate and retroviral vectors are more readily displaced by fluid flow, transduction efficiency was increased by 2-fold more than for lysate alone leading to an overall increase of 12-fold. This approach utilizing the lysate-acoustic synergy can be readily scaled-up to transduce large numbers of cells.
BIOT 4 - Self-assembly of multipotent adult progenitor cells (MAPCs) and differentiation to the hepatic lineage
Kartik Subramanian1, Karen Pauwelyn2, Catherine Verfaillie2, and Wei-Shou Hu1. (1) Department of Chemical Engineering and Materials Science, University of Minnesota, Amundson Hall , 421, Washington Ave S.E, Minneapolis, MN 55455, , (2) Stem Cell Institute, Katholieke Universiteit Leuven, Leuven, Belgium
Multipotent adult progenitor cells (MAPCs) isolated from postnatal rat, mouse, and human bone marrow can be expanded in vitro without senescence and can differentiate into cells with morphological, phenotypic, and functional characteristics of hepatocytes. Several studies have shown that spheroidal aggregate (3D) culture of primary hepatocytes resulted in the maintenance of viability and enhancement of liver specific functions over a long culture period. We thus investigated theability of MAPCs to self-assemble into 3D aggregates and explored the possibility of enhanced differentiation. MAPCs were successfully induced into 3D aggregates that exhibited good viability, morphology and differentiation potential based on expression of several endoderm markers like HNF3b, AFP, AAT, TTR and albumin. Advantages of this approach are in obtaining more functionally mature differentiated cells, as a model system for studying nascent 3D development and in designing scalable culture systems that can be monitored and controlled to enhance differentiation.
BIOT 5 -3-D differentiation model of ex vivo megakaryopoiesis
Younes Leysi-Derilou1, Nicolas Pineault2, Carl Duchesne1, Jean-François Boucher2, and Alain Garnier1. (1) Chemical engineering, Université Laval, Pouliot building, Cité Universitaire, Quebec city, QCG1K 7P4, Canada, Fax: (418) 656-5993, , (2) R&D, Héma-Québec, Quebec city, QCG1V 5C3, Canada
Megakaryopoiesis (MKpoiesis) is a complex process by which hematopoietic stem cells (HSC) differentiate progressively into megakaryocytes (MK), which are large polyploïd cells, from which blood platelets shed. To better understand this process, we have developed a new 3-D dynamic mathematical model of MKpoiesis and have applied it to in vitro platelet production from cord blood stem cells. This model is based on an eight compartment representation of MKoiesis obtained by the division of three phenotypical dimensions into two levels each (-/+), the three dimensions being: 1) CD34+ cells (HSC), 2) CD41+ cells (megakaryocytes, MK), and 3) CD42+ cells (mature MK). One differential balance equation per compartment was stated, by considering differentiation, proliferation or death fluxes effects, and assuming logistic relations for the proliferation fluxes and constant rates for the other processes. The final nine differential equation model (eight cell- plus one platelet compartment) was solved numerically, and it's parameter evaluated by non-linear regression over experimental data obtained at two different temperatures (37 and 39 °C). Finally, the regression results have been validated by assessing the parameters identifiability and confidence intervals. Sensitivity analysis was also performed to evaluate the effect of the parameters on the responses. A clear effect of temperature on the different steps of MKoiesis was observed, and the relative importance of differentiation and proliferation on the overall cell expansion was also evaluated.
BIOT 6 - P53 tumor suppressor protein affects hematopoietic stem cell differentiation in the megakaryocyte compartment
Peter G. Fuhrken1, Pani Apostolidis1, Stephan Lindsey1, William M. Miller2, and E. Terry Papoutsakis2. (1) Department of Chemical and Biological Engineering, Northwestern University, 2145 N. Sheridan Road, Evanston, IL 60208, , (2) Department of Chemical & Biological Engineering and The Interdepartmental Biological Sciences Program, Northwestern University, Evanston, IL 60208-3120
The molecular mechanisms underlying differentiation of hematopoietic stem cells (HSCs) into megakaryocytes is poorly understood. During megakaryopoiesis, committed progenitor cells undergo endomitosis resulting in polyploid, multi-lobated nuclei. Subsequently, a constitutive program of apoptosis is linked to proplatelet formation. Transformation-related protein p53 can act as a transcription factor affecting both cell cycle control and apoptosis. The up-regulation of several known p53 target genes during megakaryocyte differentiation led to a hypothesis that p53 activation is involved in regulating megakaryopoiesis. A p53-DNA binding activity assay showed increased p53 activity during megakaryocytic differentiation in a validated human model cell-line system (CHRF cells). We generated stable CHRF clones expressing microRNAs that knock down p53 expression. Phorbol-ester-induced megakaryocytic differentiation of these cells results in higher ploidy and viability than cells expressing scrambled controls. This work broadens our understanding of the p53 regulon's role in HSC differentiation and points to ways of manipulating stem cell fate in vitro.
BIOT 7 - Differentiation of adult human stem cells guided by mechano-sensing of matrix elasticity
Florian Rehfeldt, Adam J Engler, and Dennis E. Discher, Department of Chemical and Biomolecular Engineering, University of Pennsylvania, 112 Towne Building, 220 South 33rd Street, Philadelphia, PA 19104-6315, Fax: 215 573 2093,
Human mesenchymal stem cells (MSCs) from bone marrow are promising candidates for potential therapeutic applications since they can differentiate into various lineages. State of the art differentiation of these pluripotent cells is done biochemically using appropriate growth factors. But biochemical stimuli are just one part of the complex chemo-physical environment cells face in vivo. It is now well acknowledged that cells feel and respond to their physical environment and that these physical cues are as important as the biochemical ones. Recently, we demonstrated that substrate elasticity can even direct MSC differentiation to osteogenic, myogenic or neurogenic cells. While these experimental results are striking, understanding of the complex underlying molecular mechanisms of force sensing and transduction is only at its very beginning. A detailed insight to cell adhesion on tunable biomimetic substrates is given and the connection between cell adhesion, mechanical properties of the substrate and stem cell differentiation is highlighted.
SUNDAY MORNING
8:00 – 11:10 am BCEC 107 A/B
Biophysical and Biomolecular Symposium: Protein Stability
T. G. Fernandes, S. J. Kwon, M -Y. Lee, M. M. Diogo, C. Lobato da Silva, J. M. Cabral, J. S. Dordick, Organizer Papers 8-15
BIOT 8 - Use of biophysical techniques for manufacturability assessment of therapeutic proteins
Cynthia Li1, Yijia Jiang2, Juraj Svitel1, Jie Wen1, Ranjini Ramachander2, Shengwu Wang1, Jenny Li1, and Linda Narhi2. (1) Amgen, Inc, Thousand Oaks, CA 93120, , (2) Global Cellular and Analytical Resources, Amgen Inc, Thousand Oaks, CA 91320
PURPOSE: The purpose of this study was to assess the manufacturability by comparing the effect of pH, storage and stress on the conformation, thermal stability and propensity for self-association of therapeutic proteins. METHODS: CD, fluorescence, FTIR, and Raman spectroscopies, dynamic light scattering (DLS), sedimentation velocity analytical ultracentrifugation (AUC-SV) and differential scanning calorimetry (DSC) RESULTS: The conformational stabilities of therapeutic proteins at different pH with different salt concentration or under different stress and storage conditions may affect the quality of the final protein products. This report describes the effect of low pH on the conformation and thermal stability of the proteins, the reversibility of any acid-induced changes in conformation, and also compares the solubility of the proteins at neutral pH. The secondary structure was assessed by far UV CD, FTIR and Raman spectroscopy, the tertiary structure was assessed with near UV CD, Raman and fluorescence spectroscopy, the surface hydrophobicity was compared using ANS binding, the conformational and thermal stability was assessed by DSC, and self-association was analyzed by both dynamic light scattering using a Zetasizer, and sedimentation velocity analytical ultracentrifugation.
CONCLUSIONS: Changes in conformation, self-association and thermal stability of the therapeutic proteins studied were observed at pH 4 and below. The changes in secondary structure induced by acid pH are mostly reversible for all proteins. The changes in tertiary structure that occur at pH 3.5 and above are also reversible for the proteins. Small irreversible changes in the tertiary structure of the proteins occur at pH 3. Different proteins unfold to a slightly different extent at pH 4 and below. Some of the differences in stability and solubility could result in difficulties during processing, formulation, and delivery. Manufacturability assessments of the therapeutic proteins by biophysical characterization techniques prove to be very useful during early stage development.
BIOT 9 - A novel bioseparation approach: Use of chaotropic agents for HIC and ion exchange chromatography to dissociate noncovalent multimers during purification of an Fc fusion protein
Allen B. Magill II, Pharmaceutical Development, Centocor, R & D, 145 King of Prussia Road, Radnor, PA19085, Fax: 610-993-7864,
An Fc- fusion protein formed over 30% non-covalently associated multimers in cell culture harvest. Following Protein A low pH elution, nearly 95% of the protein was multimeric. The protein was converted to an active monomeric state using chaotropic agents such as guanidine HCl (GuHCl) and urea with minimal effect on biological activity. A process was developed that included an HIC step utilizing the conductivity of GuHCl for binding. Binding and eluting in GuHCl and urea, respectively, was necessary to maintain the protein in a monomeric state. The HIC step significantly reduced aggregated and degraded protein. Additionally, a polishing anion exchange step was developed using urea to maintain the monomeric state of the fusion protein. Due to urea degradation in solution to isocyanate and ammonium, several steps were implemented to maintain less than 1% carbamylation of the protein by isocyanate. Purified Fc-fusion remained monomeric once dialyzed into formulation buffer.
BIOT10 - Aggregation of a monoclonal antibody induced by adsorption to microparticle surfaces
Jared Bee1, Jennifer Stevenson2, Koustuv Chatterjee2, Erwin Freund2, John F. Carpenter3, and Theodore W. Randolph1. (1) Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO 80309, , (2) Drug Product and device development, Amgen Inc, Thousand Oaks, CA 91320, (3) Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, CO 80262
Purpose The purpose of this research was to determine the aggregation induced by adsorption of a monoclonal antibody to various microparticle surfaces relevant to the final fill, finish and storage of a biopharmaceutical product. We also investigated how formulation excipients can either aggravate or mitigate microparticle induced aggregation.
Methods Adsorption was performed by incubation of IgG (antistreptavidin, donated by Amgen Inc.) with microparticles. The suspensions were centrifuged and the protein in the supernatant quantified by UV at 280 nm or SEC and UV at 280 nm. Adsorbed and aggregated protein was then determined by mass balance.
Results The aggregation of IgG induced by adsorption was found to be strongly affected by both the surface type and the solution conditions. Tungsten microparticles caused dramatic IgG loss from solution. Addition of 0.01% Tween 20® to the buffer stabilized IgG from silica induced aggregation, but the addition of sodium chloride increased the observed aggregation. Conclusions Protein aggregation is increased in the presence of microparticles; however, solution conditions can dramatically alter the rate and extent of induced aggregation. Incubation studies with microparticles could be used for accelerated screening of formulation stability with respect to adsorption induced aggregation.
BIOT 11 - Assessment of protein stability during freeze-thaw process
Kapil Gupta1, Nitin Rathore2, Lorena Barron2, Wenchang Ji3, Feroz Jameel3, and Keith Murphy3. (1) Drug Product and Device Development, Amgen, Mail stop 30W- 3-A , one amgen center drive, Thousand oaks, CA 91320, , (2) Global Drug Product & Device Development, Amgen Inc, Thousand Oaks, CA 91320, (3) Global Drug Product & Device Development, Amgen, Thousand Oaks, CA 91320
Biopharmaceutical industries often rely on frozen storage of the bulk material due to the increased physical and chemical stability in the frozen state compared to the liquid state. However, freezing and thawing at large scale may have impact on the quality of protein products due to cryoconcentration, ice-surface induced denaturation and cold denaturation. One potential way to minimize freeze-thaw induced protein instability is by controlling the rates of the freezing and thawing processes. This study evaluates the effect of controlling the freeze rate and freeze front velocity on the product quality. In this evaluation, Celsius-pak© disposable bag technology is used for controlled freeze-thaw operation while uncontrolled freeze-thaw is performed in polycarbonate bottles.
BIOT 12 - Identifying optimal solution conditions for high concentration protein formulations: Use of self-interaction chromatography
Mark Cornell Manning1, Charles S. Henry2, Joseph J. Valente2, Robert W. Payne2, and W. William Wilson3. (1) Legacy BioDesign LLC, 1826 Monarch Circle, Loveland, CO 80538, Fax: 970-663-6006, , (2) Department of Chemistry, Colorado State University, Fort Collins, CO80523, (3) Department of Chemistry, Mississippi State University, MississippiState, MS39762
Numerous studies have now demonstrated that the osmotic second virial coefficient (B22) of macromolecules is directly correlated to solubility, viscosity, and aggregation propensity. Control of these processes is critical to developing viable high concentration formulations of peptides and proteins. Historically, B22 has been measured using static light scattering. However, such determinations are labor-, material-, and time-intensive. Furthermore, B22 values cannot be obtained for peptides by light scattering methods due to their small size. Recently, the advent of self-interaction chromatography (SIC) has allowed B22 to be obtained directly for both peptides and proteins in a rapid fashion using conventional HPLC equipment. This approach allows a number of potential formulations to be screened in a short period of time using little material. We have used SIC to identify formulations with sufficient solubility, for biotherapeutics ranging in size from peptides to monoclonal antibodies, thereby allowing product development to proceed rapidly.
BIOT 13 - Stability of high concentration rhuMAb VEGF
Hong Liu and Mary E. M. Cromwell, Early Stage Pharmaceutical Development, Genentech, Inc, 1 DNA Way 96A, South San Francisco, CA 94404,
The purpose of this study was to develop a high concentration rhuMAb VEGF formulation for subcutaneous (SC) delivery. A stability study was designed with 100mg/ml rhuMAb VEGF, based on rhuMAb VEGF's unique reversible self-association. The extent of self-association depends on pH, protein concentration and ionic strength. Formulations were tested over a narrow pH range to minimize aggregation. Protein stability in these formulations was evaluated at 2-8°C and –20°C storage by Size Exclusion HPLC and Ion Exchange HPLC. Due to concern of potential hydrolysis of sucrose under the lower pH conditions, a boronate affinity HPLC method was utilized to evaluate glycation on stability samples.