Many industries including those associated with generation of power and the manufacture of glass, steel and petrochemicals etc. have demonstrated that continuous manufacturing provides significant benefits and advantages, ranging from reduced capital and operating expenses to greater efficiency and product quality and consistency.
The traditional approach to biopharmaceutical manufacturing involves a sequence of batch unit operations separated by hold steps requiring additional tanks and/or biocontainers. Such an approach while effective is not entirely efficient since it fails to maxim ize facility use, requires large buffer volumes, and can result in overall inefficiencies and sub-optimal process economics.
An integrated, continuous approach to bioprocessing effectively connects each unit operation, minimizing the need for lengthy intermediate hold steps. Consequently, the overall process is more efficient than batch, occupies a smaller physical and environmental footprint (favorable process mass intensity, PMI, values and environmental factors), requires less initial capital investment and has lower overall operating costs. By extending the operating period (weeks or even months compared with hours to a few days) use of smaller scale equipment continuously can actually yield large quantities of product. In addition, the increased automation for continuous operations results in greater process control, more consistent product quality, minimal downtime and minimization of human intervention, reducing the likelihood of nonconformance and increased operator safety.
Development times also are often shorter for continuous than for batch-based processes.
Continuous solutions from Pall Life Sciences, for both upstream and downstream biopharmaceutical unit operations, are enabling innovative biologic drug manufacturers, Contract Research Organizations (CROs), and Contract Manufacturing Organizations (CMOs) to maximize the benefits of continuous operations. Summarized below are the individual unit operations involved in the production of biologic drugs and the single-use, continuous solutions that are readily available or are currently being developed.
Biologic Drug Manufacturing
The biopharmaceutical production process begins with translation of the biologic drug in a bioreactor using a mammalian cell culture or microbial fermentation process. The major class of biologic drugs today are Monoclonal antibodies (mAbs) which are typically produced in mammalian cells, most frequently in Chinese hamster ovary (CHO) cells. Other recombinant protein therapeutics can be produced from mammalian cell lines, microbial and insect host cell lines.
Currently the majority of current upstream processes are based on fed-batch technology although the use of perfusion bioreactors is emerging rapidly. In the present article we concentrate on the downstream processing of recombinant proteins derived from fed-batch processes. For mAb production, CHO cells in cell culture media are cultured in the bioreactor (stainless steel or disposable) and fed necessary nutrients under conditions appropriate for maximum growth. The cell culture continues for several days (typically 10–17) to a specified end point. Having reached this set point, the harvested cell culture milieu contains the recombinant protein of interest along with many unwanted materials, including both live and dead (apoptotic) cells, cell debris, nucleic acids, host cell proteins secreted by the cell as byproducts and during cell death (apoptosis), and organelles and other types of contaminants.
Step 1: Clarification
A biologic drug must first be separated from the particulate contaminants present in the cell culture prior to purification. This separation process is referred to as clarification and generates a clarified harvest cell culture fluid (HCCF). In a traditional process, the cell culture is passed through either a centrifuge or large-pore-size primary depth filter to remove a significant percentage of the solids and turbidity present. In each case there is typically a secondary polishing depth filtration step using a smaller-pore-size depth filter to achieve the target clarity needed to allow for bioburden reduction filtration prior to chromatography.
Pall secured an exclusive licensing agreement for acoustic wave separation technology from FloDesign Sonics in 2015 for continuous clarification of cell culture and introduced the Cadence Acoustic Separator (CAS) PD system in 2016. This technology has been shown to be scalable and currently a larger version of the PD system is being developed for use in cGMP manufacturing. CAS is a single-use clarification technology that enables continuous processing, which yields HCCF following polishing with a small depth filter suitable for subsequent chromatography.
Step 2: Protein A Chromatography Capture
mAbs are almost exclusively captured using a Protein A chromatography step for primary purification. In this step, the mAb initially binds to the protein A sorbent while host cell proteins and other contaminants pass through. The column is then washed and bound mAb desorbed using a low pH buffer to elute the mAb.
Other recombinant protein biologics have different chromatographic purification requirements depending on their properties but it should be noted that Pall’s continuous solutions are also applicable for those biologic drugs.
Continuous chromatography is achieved using the bench-top Cadence BioSMB PD system, the first multi-column chromatography system equipped with a single-use flowpath designed for biopharmaceutical manufacturing.
Based on countercurrent processing technology, the BioSMB technology can replace most batch chromatographic steps while maintaining existing sorbent chemistries and buffer systems. Because the columns are connected in series, the first can be loaded past product breakthrough since the second column will capture the breakthrough. The number of columns can be adjusted depending on the mAb titer of the HCCF and the desired relative flow rate. The single-use flow path provides flexibility in manufacturing configurations. Overall, the system provides improved productivity with a smaller footprint and reduced buffer tank requirements for overall cost savings since proportionally more of the column binding capacity is used compared to a batch column where loading must be terminated before product breakthrough.
Pall recommends the use of KANEKA KanCapA Protein A chromatography sorbent (from Kaneka Corporation) for the primary capture of monoclonal antibodies from clarified cell cultures. This sorbent has a high binding capacity to match current expression levels and is designed to provide high selectivity under mild conditions. It also exhibits good flow performance and alkali stability for reuse, making it ideal for continuous mAb capture processes.
The Cadence BioSMB Process system is capable of processing HCCF from bioreactors as large as 2,000 L under cGMP conditions at flow rates of up to 350L/h. The Cadence BioSMB Process system also has a single-use flow path and leverages the design established for the PD unit.
Step 3: Viral Inactivation
Following Protein A chromatography, the eluate containing the mAb is then subjected to a viral inactivation (VI) process involving treatment at low pH. This orthogonal step is conducted to ensure inactivation of enveloped viruses and typically is performed at around pH 4 for 60 minutes. It is possible to configure the Cadence BioSMB PD system to perform a VI step a continuous Cadence solution for this important downstream process that can be used in cGMP environments.
Step 4: Ion-Exchange Polishing Chromatography
Following viral inactivation (and buffer exchange if necessary), the mAb is further purified using ion-exchange chromatography. This two-step process to remove host cell contaminants and possible antibody aggregates typically involves a bind-and-elute step using a cation-exchange sorbent followed by a flow-through step using anion-exchange. However, the order of the chromatographic steps may be alternated to accommodate process conditions.
As an example a polishing platform based on anion-exchange membrane chromatography using Mustang Q in flow through which directly loads onto a cation exchange mixed mode column containing CMM HyperCel for bind-an-elute purification. During this stage, remaining host cell proteins and other contaminants, including aggregates, are effectively removed. For continuous operation, both steps can be conducted using the Cadence BioSMB technology.
Final Steps: Preparing for Final Formulation
The eluate obtained after the ion-exchange polishing chromatography operations has the high purity and high quality required for final processing and formulation. Typically just four steps remain: virus removal filtration, buffer exchange (diafiltration), concentration, and sterilizing grade filtration.
For mAbs, which are often formulated at high concentrations, diafiltration is typically performed before the final concentration step. A continuous in-line diafiltration (ILDF) to eliminate the traditionally recirculation TFF approach used for buffer exchange. By use of single pass TFF (SPTFF) technology ILDF can be accomplished in a continuous manner.
For continuous processes, biologic manufacturers can use the Cadence SPTFF technologies in various formats to achieve the target product concentration at high yield.
Continuous biopharmaceutical manufacturing is being explored by both small and large drug manufacturers. These efforts have demonstrated successful continuous unit operations and integrated lab-scale continuous processes (upstream drug production to the final sterilizing-grade filtration step).
Although individual unit continuous operations can be applied today, there is still more work to do for a fully continuous process to be implemented in manufacturing. Further development of automation/control systems as well as in-line/at-line/on-line monitoring and analysis capabilities (process analytical technology) are needed to fully realize the benefits offered by continuous manufacturing.
Even so, the systems for biopharmaceutical manufacturers and the numerous technologies under development, support innovative companies that understand the value of continuous processing.
By Peter Levison, PhD - Pall Life Sciences
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