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Overview | Short
Courses | Day 1 (Joint Session) |
Day 2 | Day 3 | Download Brochure
7:00am – 3:00pm Registration Open
7:30 Morning Coffee
Leading the Way
8:15 Chairperson’s Remarks
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Keynote Presentation
8:25 Challenges and Solutions for Biopharmaceutical Manufacturing
 Howard L. Levine, Ph.D., President and Principal Consultant, BioProcess Technology Consultants, Inc.
The biopharmaceutical industry is a dynamic one with many risks and uncertainties associated with product development and commercialization. The ability of a company to manufacture sufficient quantities of their product when they need it, whether for early stage clinical trials or to meet commercial market demands, present significant challenges throughout the lifecycle of a biologic product. Worry over “capacity crunches” resulting from notable capacity shortages for individual products in the past spurred dramatic improvements in manufacturing processes, the emergence of standardized processes and facilities for the production of biopharmaceutical products, and the massive expansion of manufacturing capacity industry-wide. This presentation will review strategies for ensuring access to adequate capacity and review future trends in biopharmaceutical manufacturing. |
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Featured Presentation
9:05 The PER.C6® Cells Technology Platform: Providing Solutions to the Manufacturing of Protein Therapeutics
 Marco
Cacciuttolo, Ph.D., Chief Executive Officer, PERCIVIA
The PER.C6® human cell line provides robust and efficient expression of recombinant proteins. This expression system has evolved into a comprehensive technology platform for the manufacturing of complex protein therapeutics. Yields of over 8 g/L in fed-batch, and over 25 g/L in the XD™ process offer unparallel advantages to the users in terms of process economics. This technology platform also includes novel approaches for product recovery and purification. The entire manufacturing process using the PER.C6® cell line results in significant savings in both cost of goods and capital expenditure. This presentation will focus on the technical aspects involved in the development of this integrated production platform. |
9:35 Systems Biology Needs Minimal Standards for Evaluating Recombinant Protein Functionality
Ario de Marco, Ph.D., Head, Biochemistry Unit, Consortium for Genomic Technologies (COGENTECH)
A constantly increasing amount of data shows that proteins can aggregate into structures of extremely different complexity. Many of these are soluble and, therefore, not immediately detectable by eye inspection. Therefore, biophysical characterization would be necessary to distinguish between native species and soluble aggregates. However, looking at the literature, it appears that such controls are not commonly undertaken when recombinant protein are produced and, in contrast, soluble fractions are considered bona fide “correctly folded proteins.” Simple quality control assays are available and may be successfully exploited for improving purification protocols and avoiding the accumulation of inconsistent results concerning protein-protein interactions. The introduction of a platform for
Minimal Information for Protein Functional Evaluation
(MIPFE) will help in material testing and data annotation and exchange among experimental labs. Furthermore, it will offer homogeneous material for large scale bioinformatic analyses.
10:05 Coffee Break in the Exhibit Hall
Achieving Higher Yield
11:00 Native HLA Protein Production in Mammalian Cell Lines
William Hildebrand, Ph.D., Chief Scientific Officer, Microbiology & Immunology, Pure Protein LLC
HLA Class I and Class II proteins regulate immune responses to viruses, bacteria, transplanted organs, transfused blood products, vaccines, and autoimmune responses. A source of native HLA proteins is needed for research, diagnostic, and therapeutic applications. We describe the development of production, purification, and QC methods for HLA class I and class II proteins in mammalian cell lines.
11:30 A New Alternative for Cost Efficient, High-Yield Transient Production of Mammalian Proteins
Volker Vogel, Ph.D., Product Manager, Product Development, amaxa - Lonza Group
In the current study we evaluated a new reagent, commercially available as "XpressNOW", for protein production in the most common HEK293 cells. The average yield of VEGF and EPO in HEK293F cells was around 25 mg/L with maximum of protein production observed at day 5 or 7 post
transfection. For a secretory 45kD growth factor protein tested in two additional HEK293 lines, 293EBNA and 293T cells, yields were at least 4 fold higher when using XpressNOW compared to another common transfection reagent. In summary, the data shows that the new reagent facilitates outstanding transient protein expression rates from HEK293F with excellent cost-efficiency. Furthermore it offered full flexibility in the choice of HEK293 cell lines and serum-free culture media, which often is an important parameter in culture and cost optimization.
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12:00 Development of a Scalable Protease Inhibition Process for
Pichia Pastoris |
Sponsored by |
Thomas Jung, M.S., Senior Director, Market Development, KBI Biopharma Inc.
In the Biopharmaceutical environment many recombinant protein products are being produced utilizing Yeast expression systems. One of the most common of the yeast expression systems, particularly for secreted biopharmaceuticals is the methylotrophic yeast,
Pichia pastoris. Commercially available protease deficient Pichia strains are frequently employed due in part to the cell’s ability to grow to very high densities, over 500 g/L, and produce recombinant proteins in excess of a gram per liter. These protease deficient strains, such as GS115, SMD1168, and KM71 possess genetic modifications to knockout or reduce host cell proteolytic activity toward the recombinant product. Such modifications ultimately serve to improve the fidelity of the secreted protein by limiting the proteolytic degradation of the protein. Therefore the use of strains lacking the genetic knock-outs could present degradation issues for the scientist desiring authentic full length secreted proteins.
This presentation will illustrate specific approaches taken to overcome such protease issues when culturing the wild type Pichia pastoris, X33 with the associated expression of a secreted recombinant protein, weighing 8.3 kDa. The initial 5L fermentation process was transferred producing 560 mg/L of secreted r-protein; however this product was entirely degraded by native protease(s). After determining by mass spectrometry that 8 amino acids were being cleaved from the N-terminus of the expressed r-protein, steps were taken to inhibit or eliminate the degradation. Three approaches were taken; 1) limit the recombinant proteins exposure to the native protease(s), 2) inactivate the native protease(s) using pH, and 3) inhibit any remaining proteolytic activity with the use of a benign substrate.
These three techniques proved to be very successful in proteolytic inhibition at the bench, but also proved to be very reproducible in large scale manufacturing. The final developed process produced an average of 350 mg/L of full length material and the proteolytic activity was reduced to non-detectable levels.
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Quality
1:45 Chairperson’s Remarks
1:50 Biological Relevance of Protein Microheterogeneity
Gregory Flynn, Ph.D., Principal Scientist, Analytical and Formulation Sciences, Amgen Inc.
Analytical testing is performed on therapeutic proteins to ensure their
potency, safety, efficacy, and product consistency. Many of these analyses
center on subtle chemical modifications found in recombinantly expressed
proteins, collectively termed microheterogeneity. These differences
result from the infidelity of biological reactions, in process degradation, and
degradation upon storage. For example, lot release and characterization
testing methods might be designed to monitor changes that result from
deamidation, oxidation, and aggregation. This talk will discuss how
monoclonal antibody (mAb) microheterogeneity, such as glycosylation and
deamidation, which might be considered critical quality attributes, are
assessed. Experiments will be described in which a mAb was isolated from
clinical serum samples to test the impact of and changes to microheterogeneity
in vivo.
2:20 QBD for Bioanalytical Method Validation
Dieter Schmalzing, Ph.D., Director, MMTech, Genentech Inc.
Quality by Design (QBD) is an area of significant industrial and regulatory activity and interest. Its main focus is the manufacturing process. This presentation discusses strategies for the possible usage of QBD for the validation of bio-analytical methods. It also addresses the advantages and challenges this approach is providing to the field of bio-analytics and how it might complement QBD for manufacturing processes.
2:50 OOS Results and the Quality of Protein Products: Lessons from 483s
Steven Kuwahara, Ph.D., Principal Consultant, GXP Biotechnology LLC
Processing factors that affect the quality of protein products will be reviewed through the use of 483s related to OOS events. The regulatory consequences and effects on product quality will be discussed by analyzing the citations. Considerations for avoiding and dealing with these events will be discussed. Possible effects on product quality will be discussed especially with respect to biologics.
| 3:20 Removing
the Log Jam from Quantification of Baculovirus Stocks
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Sponsored by |
Andrew Fosberry, Research Investigator, Biological Reagents & Assay Development, GlaxoSmithKline
Baculovirus expression systems are an invaluable tool in protein production laboratories worldwide. Titration of baculovirus stocks is therefore a crucial procedure which routinely takes up valuable experimentation time. Traditionally, the titres of a recombinant baculovirus stock have been determined by a variety of methods that include, the plaquELISA assay, endpoint dilution, and immunoassays. However, all of these methods are cost and
labor intensive; require a long processing time, and a certain degree of expertise in cell culture and virus handling. We describe efforts internally within BR&AD Harlow to reduce cycle times and provide timely titer data to support project/ program teams. In collaboration with a third party we demonstrate here a rapid and reliable indirect method for
analyzing baculovirus stocks using capillary zonal electrophoresis with evaluation/comparison against the 'gold standard' plaquELISA assay.
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3:35 Refreshment Break in Exhibit Hall
Expression System Variations
4:30 Differences in the Glycosylation of Recombinant Proteins Expressed in Different Mammalian Cell Lines
Bruno Antonsson, Ph.D., Senior Scientist, Protein Biochemistry,
Merck Serono Intl SA
Glycosylation is one of the most frequent post-translational modifications of secreted proteins and of the extracellular domains of membrane bound proteins. It has been shown to be important for the activity and the stability of proteins. Glycosylation is known to be dependent on the tissue and cell type where the protein is expressed. Most recombinant protein are not expressed in their native cell types, and thus changes in the glycosylation pattern, compared to the native protein, could effect the protein behavior. HEK and CHO cells are two frequently used mammalian expression systems for recombinant proteins. Here we examined the glycosylation of a subset of proteins expressed in these two cell lines. The proteins were expressed with a His-tag at the N-terminus and purified by metal affinity chromatography. The purified proteins were analyzed by
SDS-PAGE, isoelectric focusing and mass spectrometry. In order to further characterize the glycosylation pattern, the proteins were treated with deglycosylating enzymes. The released glucans were analyzed by mass spectrometry. Significant differences in the glycosylation of the proteins were detected between the two cell lines.
5:00 Specialized Expression Systems for Biopharmaceuticals
Peter B. Heifetz, Ph.D., President and Chief Technology Officer, OrPro Therapeutics, Inc.
A number of non-bacterial and non-mammalian cell expression systems for protein biopharmaceuticals have been developed in recent years. While only a limited number of candidates have advanced to clinical development, the results obtained to date suggest that certain applications may be most effectively addressed using such specialized expression systems. The state of the art as it pertains to plant, algal, fungal, crustacean, insect and other emerging platforms will be discussed in the context of current industry trends.
5:30 Nebula™, a Novel Escherichia coli Strain that can Promote the Formation of Correct Disulfide Bonds in the Cytoplasm
Mehmet Berkmen, Ph.D., Research Scientist, Gene Expression, New England BioLabs Inc.
Escherichia coli is ill equipped to correctly fold multi-disulfide bond proteins to high yields. Here we introduce a novel Escherichia coli strain called Nebula™ that can correctly oxidize protein within the cytoplasm. Nebula™ expresses the disulfide bond isomerase DsbC within its cytoplasm which greatly enhances the fidelity of disulfide bond formation. Expression of proteins with multiple disulfide bonds are correctly oxidized to significantly higher yields in Nebula™.
6:00 Reception in the Exhibit Hall
7:00 Close of Day
Overview | Short
Courses | Day 1 (Joint Session) |
Day 2 | Day 3 | Download Brochure
For more information, please contact:
Mary Ruberry, Conference Director
Phone: 781-972-5421
E-mail: mruberry@healthtech.com
For exhibit and sponsorship information, please contact:
Suzanne Carroll, Manager, Business
Development
Phone: 781-972-5452
E-mail: scarroll@healthtech.com
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