Abstracts

The use of depth filtration in bioprocess and fermentation applications is a well-established, widely used method for cell clarification and capture of microorganisms. Depth filters are positioned at various bioprocess stages to protect sterilizing grade filters and chromatography columns, prolonging the life and improving the efficiency of these devices. Depth filters utilize two distinct mechanisms for the effective entrapment of contaminants from fluid streams. The first is physical capture in the tortuous paths where particles are retained upon entering the depth filter matrix. The second mechanism, electrokinetic adsorption, is effective in trapping host cell derived contaminants (for example: cell debris, host cell proteins, and nucleic acids), bacteria, fungi, viruses, and other negatively charged particulates whose sizes are smaller that the nominal rating of the media. To exploit electrokinetic adsorption, a manufacturing process is used that imparts a net positive charge to the depth media that is maintained under typical process pH ranges and ionic strengths.

This presentation will detail mechanisms of contaminant removal by depth filters, effects of flux and residence time on performance, methods for producing "open" and "tight" media grades, incorporation of filter aids and other affinity materials (e.g., carbon), and technologies that optimize depth filtration performance.

Membrane Chromatography: Not Just for Viral Clearance Anymore?

Lisa Crossley, Ph.D., P.Eng.

President & CEO
Nysa Membrane Technologies

• Presentation

Resin-based chromatography has long been the workhorse of the biopharmaceutical industry, but it is not without its limitations. The transport phenomena associated with resin-based chromatography are complex, due to the need for the fluid stream to be dispersed throughout the resin bed while the solute must also diffuse into the individual pores within each bead to reach the target binding sites. Diffusion is a slow process, and the complex geometries of the pores found within resin beads provide additional resistance to the transport of the target molecule to available binding sites. Solute transport in membranes on the other hand, occurs primarily through convection, a significantly faster form of mass transfer than diffusion. Conventional membrane adsorbers offer a 100 fold increase in throughput over a comparable packed bed while achieving a similar binding capacity. In addition, recent advances in membrane technology have produced chromatographic membranes with binding capacities that significantly exceed those of their resin-based counterparts. The high dynamic capacity typical of membranes results from the predominance of convective flow which minimizes diffusion distances as compared to resin beds. This is advantageous in the purification of large molecules such as viruses, plasmids, and large proteins. This presentation will provide an overview of membrane chromatography technology and the downstream processing applications in which it is currently used, and will also examine recent advances in the field and the potential benefits to the bioprocessing industry.

Continuous Improvement in Biologicals Manufacturing: An Opportunity for Process Analytical Technology

Professor Charles L. Cooney

Department of Chemical Engineering
Massachusetts Institute of Technology

• Presentation

A challenge in biological manufacturing is continuing to improve the process while assuring safe and efficacious therapeutics for the market. There is a normal variation in biological performance, efficiency of chemical steps and human actions. However, in the drive to assure regulatory compliance and replicate the standard batch, we create a tension between learning from the variance and seeking to eliminate it. With application of new analytical technologies we can create multiple lenses on the process and develop a scientific understanding of why processes perform the way they do. One can learn how to improve the underlying fundamental operations while maintaining the integrity of product quality. The FDA's PAT initiative creates an atmosphere in which multiple analytical tools applied with an understanding of process science can be leveraged for continual process improvement. Examples from recombinant protein production will be used to illustrate the leverage of manufacturing science in improving manufacturing performance.

Scale-up Feasibility Studies on a Tangential Flow Expanded Bed Chromatography Column

Alex DiIorio, Ph.D., Director

WPI Bioprocess Technology Laboratory, Department of Biology and Biotechnology
Worcester Polytechnic Institute

• Presentation

Expanded bed adsorption (EBA) allows for the capture of proteins directly from fermentation culture broths without the need for pre-processing or clarification. By expanding the chromatography bed, insoluble particles and debris can escape the system with consequent binding of the target to the support matrix. Traditional systems utilize a distributor at the base of the column to provide a flat or near-flat velocity profile in order to improve the interaction kinetics as material passes upwards through the bed. Accumulation of material under this distributor has proven to be problematic in manufacturing settings as it cannot be completely removed during CIP and regeneration cycles. Consequently, dismantling of the column after each step or a pre-processing step to partly clarify the broth are being proposed as stop-gap measures in existing manufacturing processes, seriously compromising the original benefits of EBA. To better control the flow under the resin bed and prevent accumulation of debris, a tangential flow EBA apparatus or T-column has been designed and tested. T-column designs at the 1 and 5 cm diameter scale have been successfully tested for the capture of secreted proteins from diluted Pichia pastoris fermentation broths having cell densities on the order of 450 - 500 g/L wet weight. For a typical EBA process, culture broths are diluted with deionized water approximately 3-fold prior to introduction to the EBA column resulting in a loading condition of 150 g/L wet weight.. In this study, scale-up feasibility of a 38 cm diameter T-column was tested by comparison of theoretical plate values obtained at the 38 cm scale with plate values obtained from the already proven 5 cm design. Equivalence of plate counts between various designs under identical conditions indicates an equivalence in column hydrodynamics which is analogous to the kinetics of interaction as material passes through the column. Simulated culture broths consisting of 150 g/L fresh weight of Bakers yeast were also tested in the 38 cm column and plate counts were comparable with values obtained from the scaled down columns supporting scale-up feasibility of this design.

"A Review of Single Use Manufacturing Technologies" - Presentation

Parrish M. Galliher

Founder, President and CTO
Xcellerex

• Presentation

Incorporation of single use manufacturing technologies for both upstream and some downstream manufacturing of biologics is increasing, primarily due to reduced capital cost, set up/build out time, cycle time and operating cost. Additional advantages include increased flexibility and improved control of manufacturing quality. However, significant challenges remain in downstream manufacturing and for large scale applications, magnified by the dramatic increases in product yield achieved in cell culture in the last decade. The presentation will provide a review of single use technologies in use today and the challenges that remain for their application across the entire manufacturing process, vial to vial.

Strategies for Chromatographic Capture: Principles and Applications of Mixed-Mode Hydrophobic Interaction Chromatography and Enhanced-Diffusion Ion Exchange Chromatography

Warren Schwartz, Ph.D.

Senior Technical Director
Pall Life Sciences

• Presentation

Design of the chromatographic capture step in a purification scheme can present special challenges for the process developer. In many cases, these challenges take the form of minimizing the need for modification of feedstock composition in advance of the first chromatographic step. The need for such modification can have a significant impact on overall process economics. For example, if conventional ion exchange chromatography is employed for capture, it is frequently necessary to reduce the ionic strength of the feedstock by diafiltration or dilution. Either approach can have a significant impact on process economics. Alternatively, if conventional hydrophobic interaction chromatography (HIC) is to be used for capture, addition of significant concentrations of lyotropic salt may be required. Here, one needs to account for the cost of the lyotropic salt as well as costs for disposal of waste salt. Moreover, addition of lyotropic salt to the feedstock may lead to precipitation, requiring addition of a clarification step in advance of chromatography.

Mixed-mode hydrophobic interaction chromatography and enhanced diffusion ion exchange chromatography can provide for capture directly from feedstocks of moderate conductivity (e.g. 10 - 15 mS/cm) without need for preliminary diafiltration or dilution. Moreover, using mixed-mode hydrophobic interaction chromatography, capture is accomplished without need for addition of lyotropic salt at concentrations typically employed using conventional HIC. In many cases, there is no need for addition of binding-promoting salt. In other cases, binding is achieved at far lower salt concentrations than those employed during conventional HIC.

Three sorbents for mixed-mode hydrophobic interaction chromatography will be considered: MEP HyperCel™, PPA HyperCel™ and HEA HyperCel™. For all three sorbents, binding is driven by hydrophobic interaction, while desorption is driven by electrostatic charge repulsion. Charge repulsion is induced by adjusting pH of the mobile phase to establish like electrostatic charges on both the chromatographic ligand and bound protein. Based on this novel mechanism, one is far less dependent on increasing salt concentration to achieve binding, and far less dependent on reducing salt concentration to prompt desorption. With desorption controlled principally on the basis of pH, it is typically possible to recover the target fraction in dilute buffer, or in buffer of much lower ionic strength than that employed during conventional HIC. This characteristic can reduce or eliminate need for diafiltration or dilution in advance of the next chromatographic step.

Direct-capture from feedstocks of moderate conductivity can also be accomplished using CM Ceramic HyperD® sorbent, one of Pall BioSepra's sorbents for enhanced diffusion ion exchange chromatography. Among the Ceramic HyperD®reg; ion exchangers, CM Ceramic HyperD®reg; is particularly salt-tolerant because of the high ligand density within the "gel-in-a-shell" hydrogel. More generally, all Ceramic HyperD®reg; hyperdiffusive sorbents display reduced dependence of binding capacity on linear velocity compared with traditional macroporous ion exchangers.

During this seminar, the fundamental chromatographic mechanisms of these sorbents will be considered along with examples of their practical application.

Registration | Abstracts/Presentations | Speaker Bios