Downstream processing can be complex, expensive, and time-consuming part of biotherapeutics production. Biomanufacturers are seeking technologies to clear bottlenecks and incorporate rapid in-or at-line analytics. Data obtained from using these methods under a well established design space can then help companies better characterize, monitor, and control their processes. The BioProcess International Conference and Exhibition features a Recovery and Purification track over three days, 22–24 September 2010, that will cover these issues and provide attendees with the information needed to manage downstream challenges.
Analytical Tools for Process Monitoring and Control
Unlike the semiconductor or chemical processing industries, the bioprocessing industry is relatively new to adopting process analytical technology (PAT)-based methods for monitoring and controlling unit operations. Nonetheless, some analytical methods familiar to the industry are gaining popularity as elements for improving process understanding.
Raman spectroscopy, for example, is a quantitative technique for analyzing solutions. As an at-line or in-line analytical tool, it provides near real-time quantitation of multiple chemical characteristics of many biological solutions. Natraj Ram (senior group leader, purification, technical operations at Abbott Bioresearch Center) will present an evaluation of this technique for preparing and releasing buffers and controlling excipient levels during the formulation step (“Evaluation of Raman Spectroscopy for Purification Operations,” Wednesday, 8:15 AM).
HPLC analysis is usually conducted at production scale. The potential for variability to occur during the elution of the process columns, however, can makes it difficult for process operators to know exactly where to start and stop collecting the product pool. In his presentation, Rick Cooley (market development manager, process analytics, at Dionex Corporation) will describe how on-line HPLC-based analyzers can be used as a PAT tool to automatically control product pool collection (“Online HPLC Controlling Product Collection from Process Scale Chromatography Columns,” Wednesday, 8:45 AM).
Multivariate batch-process modeling and monitoring, particularly when coupled with soft sensors, allows operators to ensure critical process parameters stay within set specifications. As a PAT technique, it enables real-time or near real-time design space monitoring and control (supported by the FDA’s quality by design initiative). Thomas Mistretta (senior engineer, process development, at Amgen) will speak on “Using Multivariate Batch Process Monitoring and Soft Sensors for Advanced Process Control in Commercial Scale Purification Operations” (Wednesday, 9:15 AM).
Eliminating Process Bottlenecks
In some cases, high titers and cell densities are creating downstream bottlenecks, thereby delaying development time and increasing operations costs. In this session, speakers will review some current methods to overcome these challenges and reduce cycle time.
Single-pass tangential flow filtration (SPTFF) is a new technology that allows direct flow-through concentration without product recirculation, thereby streamlining downstream processing and improving capacity and yields. In the presentation “Evaluation of Single Pass TFF to Debottleneck Downstream Processing of Monoclonal Antibodies” (Thursday, 10:30 AM), Jemelle Dizon-Maspat (senior research associate at Genentech) will compare SPTFF with conventional TFF and show proof-of-concept data on reducing pool volumes with multiple monoclonal antibodies (MAb).
High-capacity and single-use technologies can help eliminate bottlenecks downstream. Blanca Lain (senior scientist, downstream process development, Percivia, LLC) will speak on how such technologies were used to manage high titers and high cell densities generated from using the PER.C6 cell line. According to Lain’s abstract, the techniques increased the process speed and flexibility and allowed the use of smaller facilities (“Downstream Breakthroughs in Downstream Processing of High Titer and Cell Density Harvests,” Thursday, 11:00 AM).
Genentech has developed two generations of facility fit models for its network comprising six production sites. These models are used to design kg/batch processes and (based on equipment and operational constraints) allow the company to identify site-specific bottlenecks and recoverable titer ranges as well as optimize processing parameters. Nuno Fontes (senior engineer and group leader at Genentech) will summarize these models in his presentation, “Process Design and Facility Fit Optimization Models for Higher Titer Purification of Monoclonal Antibodies” (Thursday, 11:30 AM).
Advancements in the Harvest Step
Particle-size Analysis: Centrifugation and filtration are common techniques conducted in cell culture harvest. The filter capacity needed depends on the size and quantity of particles that remain suspended in a broth after centrifugation. Therefore, accurate particle-size analysis is of great importance. Roy Hegedus (senior scientist, purification, process sciences, at Abbott Bioresearch Center) will discuss how particle-size data obtained during benchtop centrifuge clarification (rather than at the production scale) can be related to the capacity of the depth filters needed for clarification (“Optimization of the Harvest Step,” Thursday, 2:00 PM).
Precipitation: In his presentation, “Enabling Precipitation as an Operation to Manage Critical Contaminants in Bioprocessing” (Thursday, 2:30 PM), Daniel Bracewell (department of biochemical engineering, University College London) describes how the precipitation can be used to remove contaminants and capture product. The technique, however, is not commonly used because achieving the desired yield and purity can be challenging.
“The quest for greater process understanding combined with improved analytical capabilities means our knowledge of contaminant profiles throughout the process is increasingly detailed,” says Bracewell. “This gives the opportunity to target these contaminants particularly those found to impact process robustness.”
According to Bracewell, precipitation can be effective in removing these contaminants. However, the potentially huge experimental space (a result of the wide variety of possible precipitation reagents and conditions) presents a significant challenge. “The difficulty is further increased by the need to consider the scale-up of the solid-liquid separation techniques that follow precipitation,” he says. His presentation will discuss approaches to these issues.
Expanded Bed Adsorption Chromatography: Protein A chromatography is a common method for purifying cell cultures. Researchers are seeking alternative methods, however, that may provide better selectivity for capturing MAbs. In his presentation, Richard Wright (principal research scientist at Pfizer Biotherapeutics) will discuss a new protein A–based expanded bed adsorption chromatography media for capturing MAbs from CHO cell cultures and compare its
performance with conventional protein A chromatography (“Exploring Expanded Bed Adsorption for Capture of Antibodies from CHO Cultures,” Thursday, 3:00 PM).
BPI CONFERENCE SESSIONS
Wednesday, 22 September 2010
8:00 AM–9:45 AM: Advances in Process Monitoring and Control in Downstream Processing
Thursday, 23 September 2010
8:00 AM–9:45 AM: Plenary Session: Integration of Upstream and Downstream Processing
10:30 AM–12:00 PM: Breakthroughs to De-Bottleneck Downstream Processing
1:45 PM–3:30 PM: Implementing the Latest Tools and Techniques to Optimize the Harvest Step
4:00 PM–6:00 PM: Overcoming Challenges of Production, Purification, and Characterization of Next-Generation Antibody-Like Molecules and Protein Therapeutics
Friday, 24 September 2010
8:00 AM–9:45 AM: Evaluation and Implementation of Next-Generation Purification Technologies
10:15 AM–12:15 PM: Process Characterization for Developing Design Space
1:45 PM–3:00 PM: Utilizing Continuous Processing to Decrease Operation Time and Improve Facility Utilization
3:30 PM–5:00 PM: Applications of Automated, High-Throughput Technologies in Downstream Processing
Processes for Future Molecules
The vital bioprocessing industry is developing potential next-generation therapies. Process development engineers are already envisioning the downstream processing steps that may be needed for new classes of proteins.
For example, Diosynth Biotechnology is examining the concentration limit of a mono-PEGylated protein (protein modified with polyethylene glycol). Sarah Holtschlag (senior scientist, downstream process development, at Diosynth Biotechnology) will describe a case study demonstrating PEG was a limiting concentration factor for protein concentrations >60 mg/mL (“Investigation into the Concentration Limit of a PEGylated Protein,” Thursday, 4:00 PM).
Regenerative Medicine: Research and development scientists have taken an interest in regenerative medicine to replace or repair organs and tissue. Peter W. Wojciechowski (director of product and process development, Advanced Technologies and Regenerative Medicine, LLC) will discuss some manufacturing and analytical challenges of these products. (“Applications for Biopharmaceuticals in Regenerative Medicine,” Thursday 5:00 PM).
Vaccines: Cytos Biotechnology is developing a new class of vaccines based on virus-like particles (VLPs). These VLPs are used as carriers for target antigens. By presenting these antigens in a highly repetitive fashion, the Immunodrug platform works with the body’s immune system to trigger a targeted response. Frank Hennecke (executive vice president of product development at Cytos Biotechnology) will detail the concept, production, and application of Immunodrugs (“Immunodrugs: Development of a New Class of Therapeutic Vaccines,” Thursday, 5:30 PM).
Identifying a Design Space Using Process Characterization
Three case studies in this session will provide real-world applications of how the identification and application of the appropriate design space can facilitate process characterization while ensuring product quality.
In his presentation on “Accelerated Methionine Oxidation Due to Viral Filtration? A Case Study of the Limitations of Small Scale Models” (Friday, 10:45 AM), Tom Strickland (principal scientist, purification process development, at Amgen) describes an accelerated stability study in which small-scale filtration was performed on protein solutions. Results showed that viral-filtered samples had a higher rate of methionine oxidation compared with unfiltered samples. Researchers then conducted troubleshooting studies to determine whether the source of the accelerant of methionine oxidation is the small-scale viral filter or the bench-scale viral filtration pressure apparatus.
Interview with Bernd Rehm
Bernd Rehm (chief scientific officer, PolyBatics) will speak on “Bioengineered Protein A Polymer Beads for High-Affinity Antibody Purification” (Friday, 9:15 AM).
His presentation abstract says, “Bacterial cells were engineered to cost-effectively produce polyester beads displaying the IgG binding ZZ domain of protein A at high density. The ZZ domain is part of a fusion protein which remains naturally cross-linked to the polyester core of the beads. The performance of these beads in antibody purification and their proposed use as disposable purification media will be discussed.”
BPI: What are some of the key physical and chemical characteristics of the polyester beads that make them highly effective in antibody purification?
BR: The key characteristic of these novel polyester beads is the high-density display of functional protein A based ligands. The PolyBatics technology allows the display of highly accessible and multiple binding domains for antibodies. This leads to an exceptionally high binding capacity and affinity allowing the processing of culture supernatants with high antibody concentrations as well as short purification cycle times. This technology also enables the easy design and production of polyester bead resins tailored towards the purification of particular antibody classes or other purification needs.
BPI: What makes these beads good candidates for use as disposable purification media and how are they different from traditional protein A?
BR: The unique single-step production process of these tailor-made bioseparation resins is extremely cost-effective and easy to scale-up by using established process technologies. Hence these polyester beads can be sold at a price significantly lower than the currently established protein A resins.
The second case study involves downstream process characterization for a highly glycosylated Fc-fusion protein. Canping Jiang (senior scientist, manufacturing sciences and technology, at Bristol-Myers Squibb) will describe how analysts used the results to establish a hydrophobicity specification for the hydrophobic-interaction chromatography (HIC) resin, harvest criterion for the production bioreactor, and critical process parameters acceptance criteria for the chromatography steps (“Downstream Process Characterization for a Highly Glycosylated Fc-Fusion Protein,” Friday, 11:15 AM).
Kelby Lau (Engineer II, process development for late-stage purification at Genentech) will present the final case study in this session (“Developing and Characterizing a High Concentration Ultrafitration Process, Friday, 11:45 AM). Products with high-concentration formulations typically have complex ultrafiltration and diafiltration (UFDF) processes. Lau will discuss strategies to develop, characterize, and scale-up a UFDF process to satisfy product quality requirements, maintain project timelines, and design a process to fit different manufacturing facilities.
Continuous processing shortens product development time by eliminating the time between unit operations. Although bioprocessing remains primarily a batch-mode production, there is some discussion of transforming downstream unit operations to continuous mode.
Continuous chromatography by means of automated column switching, for example, has shown purification results comparable to those of traditional chromatography methodologies. Stephen Lyle (principal research advisor, bioprocess development, Pfizer) will discuss how the technique was conducted on both high-and low-titer harvests and exhibited yields and quality comparable with batch-mode chromatography (“Continuous Protein A Chromatography for the Purification of Monoclonal Antibodi
es Using an Automated Column Switching Approach,” Friday, 2:00 PM).
Straight-through processing (STP) enables on-demand supply of process buffer prepared in-line. The technique enables the automation of three purification steps (two chromatography steps and one filtrations step) into one continuous streamlined unit operation. Bin Lin (principal research scientist, strategic technology development, API large molecules, at Johnson & Johnson Pharmaceutical R&D) will present a case study summarizing this technique for MAb manufacturing (“Straight Through Processing (STP) in Monoclonal Antibody Purification,” Friday, 2:30 PM).
Automation and High-Throughput Methods
The incorporation of automation principles and high-throughput analysis can significantly shorten product development times and streamline downstream processing. This session includes presentations illustrating the effects of implementing automated chromatography and high-throughput screening.
Robotics-Based Chromatography: Maricel Rodriguez (senior research associate, early stage purification, at Genentech) will present “Miniaturization of Chromatography Processes for Use in High Throughput Screening” (Friday, 3:30 PM). Her research team developed robotic purification methods using a 96-well filter plate to conduct protein A affinity and ion-exchange chromatography. Results showed similar yield and product quality compared with purification using preparative columns. Rodriguez will outline other possible applications for the robotic techniques.
High-Throughput Process Development (HTPD): Screening for affinity mimetics can be a costly and time-consuming process. Incorporating HTPD techniques and design of experiments (DoE), a research team at GE Healthcare Bio-Sciences studied ligand density effects on capacity, yield, and removal of critical contaminants. In his presentation, “Multimodal Ion Exchange Resins: An Explorative Study” (Friday, 4:00 PM), Hans J. Johansson (staff scientist, R&D, GE Healthcare Bio-Sciences) will detail a study conducted on two multimodal ligands, N-benzyl-N-methyl ethanolamine and N-benzoyl-homocysteine.
High-Throughput Screening (HTS): Multimodal chromatography resins for MAb purification depend greatly on the unique processing conditions when used in the polishing step. In his presentation, Chen Wang (senior scientist II, process sciences and purification, at Abbott Bioresearch Center) describes an HTS approach to evaluate multiple mixed-mode resins under various operating conditions for flow-through polishing of a MAb molecule. Researches conducted a central composite DoE in a 96-well plate and measured key performance parameters for comparison with column run data. The selected resin was used to develop a two-column purification process, which, according to the presentation, shows product yield and quality comparable to a standard three-column process.
SPEAKER Q&A, RICK COOLEY, DIONEX CORPORATION
Rick Cooley (market development manager–process analytics, at Dionex Corporation) will talk about “On-Line HPLC as a PAT for Controlling Product Collection from Process-Scale Chromatography Columns” (Wednesday, 8:45 AM).
BPI: HPLC is a well-known technique. What makes it a good PAT tool?
RC: It’s a widely used technique in the laboratory because of its high resolution and very good sensitivity. It’s the common choice for resolving closely related molecules as well as detecting impurites at low levels. These attributes make it well suited as a PAT tool as well.
BPI: What information can be gained by placing HPLC online in the process rather than conducting the analysis in a laboratory?
RC: If you don’t have an analytical device at the end of a purification column, then it is difficult to determine when to collect the product. Rarely are these separations able to achieve a baseline separation; so you almost always have some overlap between impurities or related substances that you’re trying to remove from the product. The challenge becomes determining when to start and stop collecting the product peak that is eluting from the large-scale purification column. In many cases, this is done by collecting fractions from the column, sampling the fractions, and sending the samples to the laboratory for analysis to determine whether the fractions have the right purity to be included in the product pool. The problem with this approach is that it is not very efficient because you have to wait for assays to come back from the laboratory before you can forward process the material. Collecting these fractions takes time, labor, and there is risk of contamination during collection and handling of the fractions. If you have a satisfactory analytical tool that is connected directly to the process so it can analyze the product as it is eluting from the column, then it allows you to make a decision in near real-time.
BPI: How does HPLC compare with other potential PAT techniques?
RC: HPLC is considered the “gold standard” of analytical methods and is typically used as the reference method to calibrate the spectroscopic tools which can potentially be applied. A simple UV sensor offers some information about what is eluting from the column, but offers no specificity to aid in resolving overlapping peaks. Raman spectroscopy has some potential to resolve closely related molecules, but is not very sensitive and is frequently challenged by background fluorescence from the eluent. NIR is a PAT tool that has been widely applied in drug product manufacturing. Unfortunately, the resolution of this technique makes it difficult to resolve biomolecules whose molecular structures are very similar. The detection limit of both Raman and NIR can make quantitation of low level impurities a challenge for these technologies. The major advantages of spectroscopic-based tools are they can be applied “in-line” avoiding the need to extract a sample from the process for analysis and they can typically be operated at higher analysis frequencies. Analysis frequency is becoming much less of an issue for on-line HPLC. The use of UHPLC, monolithic columns, and dual-channel HPLC systems is making it possible to complete product purity analyses in less than two minutes in many cases.
Maribel Rios is managing editor of BioProcess International.