The Development and Application of a Monoclonal Antibody Purification Platform
The following article describes the development of a purification platform for monoclonal antibodies (MAbs) at Pfizer's Global Biologics division, including how the individual unit operations have been affected by increasing cell culture titers and subsequent changes in the impurity profile, as well as viral clearance issues. The platform was developed to maximize the efficiency of the purification process and product purity while minimizing the development time for early-phase therapeutic antibodies. The success of this platform in its various stages has been demonstrated for several MAbs representing different IgG classes produced in different cell lines.
Early Platform Description
After cation exchange, diafiltration was performed to prepare the product for anion exchange chromatography in flow through mode, in which impurities such as DNA and some HCPs were captured on the resin (and later removed by regeneration and cleaning steps). After anion exchange, the flow-through fraction containing the antibody was passed through a nanofiltration unit to remove potential viruses and then formulated in buffer in a final concentration and diafiltration step.
Early Platform Modifications
The original process was developed at Pfizer for a 500-L scale bioreactor. To accommodate an expanding pipeline, the scale of clinical manufacture was increased to 1,200 L, enabling the production of sufficient supplies in a shorter campaign. This increase in scale made it necessary to introduce changes into the platform process. One change reflected the efficiency of the initial depth filtration operation to remove cells and debris. During scale-up, we noted that effective filtration required a much larger membrane area than was feasible, so we introduced a continuous flow centrifugation process, followed by depth filtration to minimize membrane area, reduce costs, and increase the efficiency of cell removal.5
To increase process efficiency, we also changed the order of the polishing chromatography steps, allowing the intermediate diafiltration step to be removed and thus reducing the total number of unit operations. Using this process, the pH of the Protein A affinity chromatography eluate was adjusted upward after pH inactivation and loaded directly onto the anion exchange resin. The anion exchange flow-through pool was then adjusted down to an optimal pH for the subsequent cation exchange step. All other unit operations remained the same. The resulting process is shown in Figure 1 as process 2.
In the next iteration of the process, further modifications were made to process 2 to reduce the clinical manufacturing time. The cation exchange polishing step was removed and the anion exchange chromatography step was changed from a resin-based operation to one involving membrane adsorbers. This increased the loading capacity of this step by a factor of up to 100. All other unit operations remained the same. The resulting process is shown in Figure 1 as process 3.
Then, the timeline for processes 2 and 3 was reduced by 30% (Figure 2). This was accomplished by performing previously defined experiments for each unit operation with specified ranges for buffer systems, load capacities, and pH/conductivity values. These experiments allowed ~80% of the process to be determined, with the remainder defined through additional bench-scale experiments specific to the antibody product. Subsequently, the experimental conditions promoting maximum yield and product purity were used to establish the parameters for each unit operation in the platform process. After the purification scheme was established, a larger demonstration run was carried out to evaluate scale-up effects before transferring the process to the pilot plant for manufacturing clinical supplies.
Current Platform Modifications
Although process 3 worked well for many Pfizer antibodies, the performance was not satisfactory for all antibodies, particularly those produced at high titers with correspondingly high levels of impurities. In these cases, there were occasional issues surrounding virus clearance, raising safety concerns especially for antibodies required in large doses. To address these issues and increase the robustness of the platform, a cation exchange chromatography step was added to process 3 to define the current process (Figure 1). Based on knowledge gained from the implementation of the original process, and modified processes 2 and 3, the load capacities, buffer solutions, and other operational parameters were standardized to maximize purification efficiency for the majority of the antibodies in Pfizer's pipeline.
These changes reduced the timelines for purification development by 50% compared to the original process. The defined, optimized platform process has been tested at the bench scale to verify that it is suitable for the production of clinical material. At this point, it must meet yield and purity specifications before it is scaled up for the demonstration run, or transferred to the pilot plant. This process is designed to meet the purification needs for 80% of the antibody products in our pipeline, which means the majority of the purification processes can be verified without further development. The remaining antibody projects may need slight modifications to meet individual specifications. These modifications will be evaluated on a case-by-case basis.
The current process, as shown in Figure 1, provides Pfizer with a combination of purification and virus clearance robustness, allowing us to quickly move products into the pilot plant for clinical manufacturing, thus accelerating progress toward the clinic. Even though the other processes shown in Figure 1 also met these criteria, they did not meet our new standard for robustness established by increasing product titers. A process was needed that was more adept at handling changes in the composition of the cell culture broth as well as unexpected increases in titer following scale-up, requiring additional column capacities. Although the current process bucks the trend of reducing the number of unit operations, it increases the chance of meeting the purification needs of a greater number of projects in a shorter time frame. These early-phase purification processes can be optimized further once proof of concept has been achieved.
As cell culture titers continue to increase, the biopharmaceuticals industry will be faced with new challenges, including greater product heterogeneity and increasing impurity levels. As scale increases for early-phase manufacturing, resin capacity must increase to minimize operating costs, and therefore, it will be necessary to carry out studies to determine the impact of these changes on virus clearance and the removal of impurities. If increased capacity cannot meet these needs, alternative separation methods such as simulated moving bed chromatography will become paramount.
It also will become necessary to increase the throughput of nanofiltration devices, so that they do not become the next process bottleneck. In addition, we must develop new technologies for ultrafiltration to meet the need for high-concentration drugs used for subcutaneous injection. These are just some of the challenges facing the industry today.
The antibody purification platform process used in Pfizer's Global Biologics organization has undergone changes over the years in response to increases in both production scale and cell culture titers. These changes have resulted in a purification scheme that takes advantage of previous knowledge to reduce development time, while ensuring that product purity and virus clearance are sufficient for clinical manufacture. The goal is to manufacture early-phase material without having to perform downstream development, and the current process as outlined in Figure 1 is capable of meeting that goal. However, further increases in titer will likely bring more challenges, and thus it will be necessary to implement additional modifications to our process to keep pace with increasing upstream productivity.
We would like to acknowledge the entire purification group in Global Biologics for their help, as well as the cell culture groups for their provision of the cell culture broth necessary to complete these studies. In addition, these studies would not have been possible without the support of the process development analytics group.
JUDY GLYNN is senior principal scientist, TIMOTHY HAGERTY is scientist, TIMOTHY PABST, PhD, is senior scientist, GOPINATH ANNATHUR is senior scientist, KRISTIN THOMAS is senior scientist, PAUL JOHNSON is scientist, and NATARAJAN RAMASUBRAMANYAN, PhD, is an associate research fellow, and PAUL MENSAH, PhD, is an associate research fellow, all at Pfizer Global Research and Development Global Biologics, Bioprocess R&D, Chesterfield,
MO, 636.247.6519, email@example.com
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