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A purification scheme to maximize the efficiency of the purification process and product purity while minimizing the development time for early-phase therapeutic antibodies.
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.
Recent advances in mammalian cell culture technology have resulted in significant increases in upstream productivity, with antibody titers of >5 g/L becoming increasingly common. Such increases could change the characteristics of the cell culture broth, leading to higher levels of product-related heterogeneous proteins (aggregates, degradation products, processing variants) and cell-derived impurities such as host cell protein (HCP) and cellular DNA. In response, platform purification processes for MAbs, i.e., those designed to move a product through Phase 1 clinical trials with minimum development, have become more streamlined in their design and execution. Such platforms must accommodate the majority of antibodies and yet be flexible enough to cover most contingencies and cell culture or antibody-related processing issues. They must also save time and thus accelerate the progress of antibodies toward the clinic.
The MAb purification platform used by Pfizer for early-phase clinical supplies has evolved over the last five years to accommodate increases in titer and associated issues, as well as unique challenges directly related to our own cell lines and upstream processes. This purification platform has been developed to accommodate antibodies derived from both NS0 and Chinese hamster ovary (CHO) cell lines. The original process, shown in Figure 1, followed a traditional antibody purification scheme.1–4 It consisted of three major chromatography steps with two additional unit operations designed to remove or inactivate viruses. After harvest, the cells were removed from the cell culture broth by depth filtration. In some cases, the broth was concentrated before the first chromatography step because of facility fit issues. Protein A chromatography was used in the initial capture step, where the antibody in cell culture broth was loaded at neutral pH, washed with various solutions, and eluted at a lower pH value. The eluate was then held at this low pH to inactivate viruses, and then adjusted to increase the pH before the cation exchange chromatography step.
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.
The relative purification development timeline for this original process is shown in Figure 2. Initially, several range-finding experiments were carried out to determine the optimal parameters for each unit operation and to maximize the yield and purity for that particular antibody. We used these results to determine the purification scheme, which was performed in its entirety at the bench scale before a larger demonstration run. After the demonstration run, this process was transferred to the pilot plant for manufacturing clinical supplies.
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.
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.
Table 1 shows the impurity levels for various Pfizer antibodies using the processes outlined in Figure 1. The impurity profiles are collected after the final chromatography step. The percent monomer is determined by size exclusion high performance liquid chromatography, using two G3000 SWXL columns (Tosoh, Montgomeryville, PA) in series with detection of the antibody at 214 nm. HCP is detected using a high-sensitivity sandwich Enzyme-Linked ImmunoSorbent Assay (ELISA) kit specific for either CHO or NS0 proteins following the manufacturer's instructions (Cygnus Technologies, Southport, NC). We measured residual recombinant protein A levels using a sandwich ELISA kit from Repligen (Waltham, MA). Finally, residual total DNA was evaluated by extraction using the DNA extractor kit from Wako (Richmond, VA), followed by detection of the single-stranded DNA using a threshold kit (Molecular Devices, Sunnyvale, CA).
The results indicate that for the representative antibodies shown for the different process permutations, the current purification scheme as outlined is sufficient to produce an acceptable product. A few of the antibodies required minor changes to the platform process to produce the desired product purity, as explained in Table 1. Figure 3 shows the overall virus reduction achieved for the early-phase representative antibodies in the different platform processes. Again, the results indicate that all processes achieved satisfactory viral clearance. However, the current process is advantageous as we increase column capacities to accommodate increasing titers, as insurance for acceptable virus clearance results.
Table 1. Impurity profiles and classifications for various therapeutic monoclonal antibodies using the platform processes outlined in this article
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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