Enhancing Manufacturing Development Efficiency

Svetlana Dukleska (Merck)

Early Research Stability Studies Can Aid in Rapid Screening of Potential Drug Candidates

In a competitive research and development (R&D) space with tight time lines, it is important to have a toolbox of extended characterization and analytical assays for rapid screening of potential candidates. Additionally, early research stability studies can provide initial information on potential degradation pathways for screening candidates, for purification development, and for establishing stable formulations.

Herein, we show the development assessment of an IgG2 monoclonal antibody (MAb) using degradation pathways that were determined by early research stability studies and extended characterization of the molecule. Cation-exchange high-performance liquid chromatography (HPLC) analysis of material stored at elevated temperatures showed a severe shift toward acidic variants after a one-month storage. Liquid chromatography with mass spectrometry (LC/MS) analysis of temperature-stressed samples showed that the molecule was prone to deamidation and isomerization in its light chain. The shift towards acidic variants correlated to the level of deamidation and isomerization as evaluated by LC/MS. We determined that the cation-exchange HPLC assay can be used as a quick turn-around assay to screen potential clones for candidate development. The early research stability study combined with extended characterization and analytical evaluation to provide information on degradation pathways that aided in efficient candidate clone selection, purification, and formulation development.

Dogan Ornek (Lonza Biologics Inc.)

Characterization of a Biomanufacturing Fermentation Process Using a New DoE Approach: Definitive Screening Designs

Traditionally, a bioprocess is characterized using a two-step approach that involves factor screening followed by response surface designs. That approach is very costly and time-consuming because it requires a significant number of individual experiments. In 2011, Jones and Nachtscheim described a more efficient design that allows for factor screening, resolution of two-way interactions, and estimation of quadratic effects in a single-step. The Definitive Screening (DS) design has not yet been validated on a bioprocess.

Here, we compare traditional and DS design-of-experiment (DoE) approaches to characterize a pDNA fermentation process. A 15-run DS design was evaluated against a 31-run fractional factorial design augmented with axial points to accommodate quadratic process effects. The designs included as experimental factors pH, %DO, induction temperature, induction OD₆₀₀, and feed rate for a total of five input parameters. We used volumetric pDNA titer as the output. Both designs resulted in equivalent process characterization and modeling. Main effects, two-factor interactions, and quadratic effects were comparable in both design models. The traditional approach required 31 runs whereas the DS approach required 15 runs. So our comparison validates the use of DS designs for fermentation process characterization, with a substantial cost and time savings over traditional DoE experimental approaches. Details of our comparison and model construction will be covered.

Jennifer Weber (Corning Incorporated)

Human Mesenchymal Stem Cell Attachment and Expansion on Synthetic Corning Microcarriers in Serum-Free Defined Media

Cell-based therapies require efficient culture of cells in large quantities.

A three-dimensional environment provided by microcarriers — rather than traditional two-dimensional methods — enables rapid cell expansion in dynamic conditions. Current commercially available microcarriers generally require time-consuming and labor-intensive preparation (e.g., preswelling in water or buffer, autoclave sanitization, and coating with extracellular matrix proteins) before cell seeding. Further, serum-containing medium may be required to facilitate cell attachment and expansion in stirred conditions.

Such limitations hinder the use of microcarriers both for cellular therapeutics and biopharmaceutical manufacturing. Here, we report on expansion of human-bone-marrow–derived mesenchymal stem cells on synthetic microcarriers with defined, xeno-free medium. Cells were maintained for multiple passages on gamma-sterilized, ready-to-use Corning microcarriers in spinner flasks. Further, we demonstrate cell expansion by bead-to-bead transfer without the need for enzyme dissociation.

Cell Culture and Upstream Processing

Gayatri Ramasamy (Loughborough University)

Optimization and Automation of Umbilical-Cord–Derived Mesenchymal Stem Cell Culture for Clinical Applications

Optimization and automation of umbilical-cord–derived mesenchymal stem cell culture for clinical applications. Mesenchymal stem cells (MSCs) present a promising potential for regenerative medicine applications because of their potential to differentiate into lineages of mesoderm origin. Bone marrow is the main source of MSCs; however, aspirating bone marrow from donors is invasive, donors are limited, and proliferative capacity decreases with increasing donor age. Umbilical cords (UCs) are an excellent alternative source of MSCs because UC cells are younger and can be cryopreserved and stored in stem-cell bank for donors and their families. In addition, UC cells have low immunogenicity and better tolerate HLA mismatch.

Following isolation, MSCs will require in vitro expansion to provide sufficient functional biological material for a cell-based therapy. Currently, most stem cell culture processes are conducted at laboratory bench scale by manual operation. Automation of such processes will increase standardization, reduce uncertainty by reducing variability, and ultimately lower costs. The aim of my research is to optimize and automate a manual cell culture process of umbilical-cord–derived MSCs to prove the potential of such technology in clinical applications. Various culture conditions have been investigated, including xeno- and serum-free culture conditions.

Recovery and Purification

Maybelle Woo (EMD Millipore)

Considerations and Challenges in Preuse Sanitization of Depth-Filtration Media

Clarification of cell cultures by depth filtration has long been a well-accepted technology. Depth filters are relatively easy to install, with fairly straightforward clean-up. Not much emphasis has been placed on the cleanliness of depth filters before their use. As customers begin working with higher-titer feed stocks, more robust processing, and single-use processes, reduction of bioburden is increasingly a concern. Although the clarification step typically is followed by a sterile filter that should be a catch-all for trace contamination, undue stress on the sterile filter can very easily result in plugging, which is typically resolved with increased sterile filter surface area. At large scales, additional sterile filter area can quickly become very expensive. Preuse sanitization or sterilization of depth filters used for clarification can help with bioburden reduction. However, cleaner media with reduced bioburden does come at a price.

We examined five different methods of sterilization/sanitization: autoclaving, caustic sanitization, gamma irradiation, ethylene oxide treatment, and chlorine dioxide treatment. This poster highlights process trade-offs associated with each method. The underlying trend is that performance is unaffected. But ultimately, to clean or not to clean should be decided by users for their specific applications and process needs.

Donna Quicho (Genentech, Inc.)

High-Throughput Resin Screening to Support Process Development of Bispecific Antibody

Aided by the Tecan robot, high-throughput screening (HTS) of resins can be a very useful during downst
ream process development for novel proteins. In this case-study, we use HTS during development of the purification process for bispecific antibodies. Those are produced by annealing two different half antibodies that contain heavy and light chains and are expressed in Escherichia coli. The annealing process produces several product-related impurities that have characteristics very similar to those of the target bispecific antibody, so they are very challenging to remove in downstream purification.

To improve process development time and resources, we used HTS. Multiple pH and salt conditions were evaluated for several different resins to identify suitable conditions that separated the bispecific antibody from its impurities. Identified resins and conditions were tested and optimized using a packed-bed column. This presentation will discuss the predictability between the HTS and packed-bed column behavior.

Michael Thomas (Asahi Kasei Bioprocess)

Comparison of Hollow-Fiber Microfilter Operation Methods for Mammalian Cell Perfusion Culture

Perfusion of mammalian cell culture requires a gentle device with high retention efficiency, high capacity, and the ability to perform consistently during lengthy filtrations. In addition, high-protein transmission and a clarified perfusate are desirable. Hollow-fiber membranes are widely adopted in perfusion culture cell separations because of their clarification ability; however, there sometimes are concerns about protein transmission. We explore the use of traditional tangential-flow filtration (TFF) mode and alternating tangential-flow (ATF) mode for cell separations. Both membrane type and operational methods are compared in terms of protein transmission.

John Fisher (Asahi Kasei Bioprocess)

Protein Adsorption and Impurity Removal Characteristics of Anion-Exchange Hollow-Fiber Membrane Adsorber with Grafted Chains

Membrane adsorbers are widely recognized as powerful tools for the purification of biopharmaceutical protein products. Here we study a membrane adsorber based on a 0.2-μm polyethylene membrane with unique, long, grafted chains that have weak anion-exchange ligands attached. Those structural characteristics make this membrane adsorber an effective tool for protein purification processes.

Host-cell protein reduction is investigated over a range of buffer conditions. Two-dimensional gel electrophoresis analysis reveals that a wider range of molecular weights in the low-pI population of proteins are removed versus anion exchange resins. Robust removal of DNA is observed. Using bind and elute conditions, it is possible to capture and collect higher molecular–weight-protein targets, more effectively than existing anion-exchange resins and membrane adsorbers.

Nripen Singh (EMD Millipore)

Next-Generation Clarisolve Depth Filters for Clarification of Flocculated Mammalian Cell Culture System Feeds

Increasing bioreactor cell densities and decreasing cell viabilities at harvest have posed a significant challenge to cell culture clarification operations. Traditionally, separation technologies such as centrifugation and filtration techniques (tangential-flow filtration and depth filtration) have been widely used for clarifying high–cell-density cell culture broths. However, the increased cellular debris present in such complex feed streams can prematurely foul membrane and commercially available depth filters, adversely affecting their capacity and throughput. Flocculation of cell culture harvests by chemical means (using either acids or polymers) has been used as an alternative separation technology to enhance the clarification throughput and downstream filtration operations. Although flocculation is quite effective in agglomerating cell debris and some contaminants (e.g., host-cell proteins and DNA), the resulting flocculated suspension is generally not easily separable by ordinary filtration methods.

In this work, EMD Millipore has developed next-generation Clarisolve depth filters that, when coupled with process optimization, improve primary and secondary clarification for flocculated high–cell-density mammalian cell culture system feeds. The design of the filter media and the interactions between the flocculation method and the filter will be discussed.

This is just a sample of the posters that will be on display in the exhibit hall. These early arrivals will be joined by more in their categories, as well as others in Product Lifecycle Management, Antibody–Drug Conjugates, Analytical Advances, Viral Safety, and Vaccine Production. Posters provide cutting-edge science, techniques, and expertise to improve existing methods and processes while reducing time and cost. BioProcess International (in partnership with the BPI Conference) is proud to present the Third Annual Best Poster Award at the 2012 BioProcess International Conference and Exhibition. Posters will be reviewed and judged by editor-in-chief Anne Montgomery and members of her Editorial Advisory Board. Two winning posters — one academic/industry and one supplier — will be announced at the conference and published in BPI’s November 2012 Interactive Poster Hall supplement as well as on the BPIMobile app. Posters, complete with prerecorded audiocast presentation, will also be posted online for a full year at bioprocessintl.com/posters.