K. John John Morrow Jr. Ph.D. President Newport Biotech
Sartorius Stedim Biotech Forum Focuses On Next Level Bioprocess Operations
In the early 21st century the bioprocessing industry fretted about a production crisis that never happened. Concern arose a decade ago that downstream purification facilities would not be adequate to meet anticipated increases in cell culture titer from upstream fed-batch bioreactors.
In fact, for monoclonal antibody platforms, the mean bioreactor cell culture titer for most CHO cell lines of interest to the industry continues to be in the range 1.5 to 2.5g/L, well within the capability of current downstream platforms. Today, the focus upstream has shifted to increasing productivity through continuous processing via single-use perfusion bioreactors. The challenge for the downstream phase is to develop unit operations that are able to handle the continuous flow of material from these bioreactors.
At the recent Sartorius Stedim Biotech (SSB) Upstream and Downstream Technology Forum in Foster City, CA, speakers dealt with both sides of these technological challenges. Parviz Shamlou, Ph.D., of the Keck Graduate Institute, set the stage for the presentations to follow.
“First-generation biopharmaceuticals suffered from low efficiency, significant hidden costs, and substantial regulatory oversight,” he said.
“There have been significant improvements with second-generation biologics including monoclonal antibodies, but cost and development time continue to limit progress.”
Dr. Shamlou cited a lack of process and product knowledge as the root causes hindering more efficient and economic new biotherapeutic development.
“First-generation processing is based on data from trial and error experimentation,” he explained. “We need to move to principles based on a thorough mechanistic understanding. This entails quality by design, using Six Sigma processing. These strategies have significantly less hidden costs, higher overall yields, higher productivity, and the potential for less regulatory oversight.”
Dr. Shamlou has investigated the challenges of high-viscosity formulations of biologics. This is an especially significant problem for patients and a challenge for process development. For example, the fact that these drugs ideally need to be administered subcutaneously (SQ) through injection places considerable demands on the formulation process. Patients favor small needles for drug delivery and demand injection times of a few seconds.
Critically, the volume is typically limited to 1–2 mL and, for most patients, the injection force (glide force) should be as low as possible (8–10 Newtons). All this means an upper limit to formulation viscosity of approximately 5–8 cP (centipoises, the standard measurement of viscosity) corresponding to a mAb concentration of approximately 120–150 mg/mL. The challenge is that this concentration is often below the typical dose required for treatment. Currently, there is no solution to creating clinically acceptable protein formulations that can mitigate these issues.
From a processing perspective, high-viscosity protein solutions are especially relevant to tangential flow filtration (TFF), which is the unit operation of choice for concentration and buffer exchange. Fouling of TFF filters and loss of flux is a particularly difficult issue that remains to be addressed.
Dr. Shamlou further described the use of an alternating tangential flow-assisted perfusion bioreactor for protein harvest. The device employs a single-use hollow fiber filter module which is powered by a cycling pump driving a diaphragm up and down every 10 to 20 seconds. The ATF™ system from Refine Technology was originally designed for perfusion processes in mammalian cell culture, using hollow fiber filters to achieve an efficient cell separation with low shear and to allow robust large-scale manufacturing.
Product retention is a challenge as Dr. Shamlou outlined the use of computational fluid dynamics (CFD) to describe and simulate the complex flow in the ATF operation. He emphasized that CFD is one of several scale-down tools that allow bioengineers to understand the interaction between process parameters and product quality during processing.
Scale Up and Scale Down
With tangential flow filtration of protein solutions, researchers are able to concentrate and buffer exchange biotherapeutics to their proper dose and excipients, according to David Briggs, Ph.D., a research scientist in manufacturing sciences and technology at Avid Bioservices. “However, we recognize that at higher protein concentrations this may present a problem, and of course the future of the industry will focus on highly concentrated protein solutions,” he pointed out.
Dr. Briggs discussed his experiences in fine-tuning TFF, which is also known as cross-flow filtration. The technology is based on running the field stream parallel to the filter device. Small molecules are eliminated, and the larger ones are concentrated in the retentate. While TFF can be employed anywhere in the protein purification process, in general it is only used in the final formulation step.
Dr. Briggs uses diafiltration (DF) in the purification process, by which new buffer is constantly introduced into the flow. With the dilution of the solutes the unwanted molecular species and residual starting buffer components are gradually removed. “During DF, keeping the retentate volume constant is necessary for efficiency and reproducibility,” he told the forum attendees.
A major feature of TFF is that it can be upscaled and downscaled to meet the demands of different protocols without adversely affecting the parameters of the process. The data establish that at large and small scale, flux rates were similar during concentration and that step yields and recovery rates during the entire TFF operation were similar. Finally, at both the manufacturing scale and at lab bench scale, conductivity decreases as a function of diavolumes were similar.
Virus Safety Assurance
A major axiom in bioprocessing is that you can never completely rule out viral contamination. So stated Hazel Aranha, Ph.D., field marketing manager at SSB. “But we do have several robust virus clearance technologies available that allow a strong level of confidence,” she added.
While there are both upstream and downstream methodologies available, the downstream component is a regulatory requirement whereas upstream is addressed as essentially a business insurance consideration. “These concerns are driven by the recognition that biologicals are life-saving medications and risks associated with these biotherapeutics must be stringently managed and monitored,” explained Dr. Aranha.
As it happens, a viral contaminant in a biopharmaceutical has never ended up in the final product and iatrogenically transmitted to the patient, claimed Dr. Aranha. “However they have been detected in bioreactors,” she said. “This excellent safety record can mainly be attributed to the ‘viral safety tripod’: regulatory guidance that recommends a holistic approach, which is strictly adhered to within the industry; adequate sourcing, documentation of virus clearance evaluation; and in-process testing.”
Industry practices, in general, have proven to be adequate at the upstream end, but it is important to be aware of the most at-risk entry points for contamination. “Most cells today are grown using serum-free products but serum free doesn’t mean animal-derived material free,” continued Dr. Aranha. “Evaluation of components from animals is one critical place where you have to ask the right questions, and viruses can also gain entry to the product and manufacturing environments via raw materials, purification reagents, and excipients.”
Today the industry is much more open about difficult issues than it was in the past, and problems that would not have surfaced are now being discussed, as Dr. Aranha has described in her writings: “You may lose face by admitting that you have a problem, but in the long run openness is the best strategy for dealing with these crises.”
Where is the industry headed? There are several exquisitely sensitive methods for detecting contamination, including massive parallel sequencing, degenerative polymerase chain reaction, mass spectrometry, and panmicrobial microarrays. However, all these approaches have their shortcomings and their consideration for routine monitoring must be tempered with caution.
While PCR-based detection methods are accepted detection methods, viral infectivity assays are still widely used and are not obsolete.
“We need more risk mitigation upstream,” concluded Dr. Aranha. “Fortunately for us, downstream is pretty well controlled at this time. But we have to recognize that the level of detection that we strive for is a balance between what is feasible and what is workable and cost-effective.”
What are the benefits of single-use technologies at the downstream end of contract manufacturing and do they outweigh their shortcomings? According to John Moscariello, Ph.D., of CMC Biologics, “At the upstream end, the advantages are clear: reduced plant size because of the smaller footprint, reduction in bottlenecks, increased throughput, and operating cost savings,” he explained. “However, the implications are not as easy to quantify for the downstream end, as this is often not a rate-limiting step for plant throughput. So the implications for turnaround time, infrastructure reduction, and capital cost savings are less.”
Nonetheless, there is renewed interest in single-use systems in downstream processing as the company begins to pursue continuous processing. Some of the options under consideration are disposable centrifugation, pre-packed disposable columns, single-use mixers and single-use tangential flow filtration. “There are reasons that disposable downstream processes have lagged behind, mainly related to the fact that the downstream footprint is largely a function of mass to be processed, not volume,” he noted. “The advances in cell culture development have enabled the production of similar mass in smaller volumes that enabled disposables in upstream processes.”
Dr. Moscariello presented several examples demonstrating the advantages of disposable options. “Because of significantly higher loading, disposable membrane chromatography modules become much more desirable with smaller lot size,” he said.
Since single-use, by definition, precludes reuse, there is no disposable option for large commercial demands because single-use would not be cost effective. However, there is still a significant benefit for early clinical programs, where molecule attrition is high and there is a desire to show proof of concept in the clinic rapidly and with minimal expense.
“We believe that disposable chromatographic options can be cost-effective, but a rigorous economic analysis must occur for each process, said Dr. Moscariello. “This includes considerations not only of volume size, but also loss of resin due to expiry date (shelf life), number of cycles that the resin can endure, buffer utilization and validation, and supply chain issues.”
Augmented Reality Training Tool
During the forum Frank Maggiore, project engineer at SSB North America, brought up the topic of an augmented reality (AR) training tool. What is it? The answer is more obvious than most people realize.
“Augmented reality” or the superimposing of graphics or data over real-world environments is well-known to every football fan. TV screens are filled with superimposed images during the game, such as the first-down marker line. This technology applied to the biotech industry has a wealth of useful applications,” said Maggiore.
AR can be achieved using a computer, software, cameras, visual displays, and multiple marker types to provide recognition and positional information. SSB has developed a prototype AR training tool application which runs on an iPad. It provides guided training that takes the viewer through the entire process of assembly and operation of a single-use Biostat® STR bioreactor.
Maggiore discussed a Boeing study that used a similar augmented reality training tool for a mock wing assembly exercise where the performance of the AR tool was found to be superior in comparison to conventional desktop and mobile instructions.
Because human error is responsible for much of the failure in biotech processes, AR reality can provide improved training and risk mitigation saving time, money, product loss, and batch failures, explained Maggiore.