Implementing a Single-Use TFF System in a cGMP Biomanufacturing Facility

For a multiproduct cGMP manufacturing facility, measures to prevent risk of cross-contamination between products are a high priority. Single-use technology can provide a simple method of achieving this goal for many processing operations (1). This case study describes how the transition from multi-use to single-use tangential flow filtration (TFF) was evaluated and implemented for a buffer exchange step in a 500-L production-scale recombinant protein manufacturing facility (PF1) at CMC Biologics, a contract manufacturing organization (CMO).

A typical monoclonal antibody (mAb) or recombinant protein manufacturing process uses ultrafiltration/diafiltration (UF/DF) by TFF for final product concentration and formulation of bulk drug substance (see Figure 1A). The primary criteria for such a TFF system are suitability for cGMP, product compatibility, and reliable performance, including high yield and efficient buffer exchange. Additional criteria of almost equal importance, particularly for a CMO, include rapid and predictable setup and operation, a platform system with multiple filter options to accommodate a wide range of protein products, and low total operating costs, including materials, labor, buffer usage, and overhead due to changeover between products. These last criteria are particularly well addressed by single-use technology. A primary objective of single-use systems is to simplify and reduce costs associated with product changeover, because cleaning and cleaning validation requirements are significantly reduced when product-contact surfaces are discarded after use. Additionally, for TFF, total operating time in routine usage is reduced through elimination of many of the steps required to clean and store membranes between manufacturing batches (see Figure 1B). As re-usable TFF membranes are typically product-dedicated, the significant logistics of storing and tracking multiple use TFF membranes for different protein products between manufacturing campaigns is completely eliminated by a single-use system. The economics of single-use TFF technology are also favorable relative to re-usable membranes for intermediate-scale manufacturing with relatively few batches in a campaign (2).

Figure 1: Process flow diagrams. (A) A typical process flow for a monoclonal antibody or recombinant protein. Ultrafiltration and diafiltration (UF/DF) for buffer exchange is performed at the end of the manufacturing process for formulation of bulk drug substance. (B) Processing steps for UF/DF by tangential flow filtration (TFF). Steps that differ between re-useable and single-use filters are indicated on the right. Brackets on the left indicate processing times for pre- and post-use operations.

The PF1 cGMP manufacturing facility at CMC Biologics was designed for mammalian cell-culture processes up to 500-L production scale based on single-use technology. Typical PF1 clients have clinical-stage protein therapeutics with a need for relatively few (e.g., two to five) batches in a manufacturing campaign. Thus, between frequent needs for product changeover and favorable economics for short campaigns, a single-use TFF system was an obvious fit. However, to streamline facility commissioning, single-use technology was initially emphasized more heavily in upstream production and buffer preparation, allowing many of the downstream purification operations to leverage existing equipment and methodology from the re-useable systems already established in a parallel 3000-L production facility. Once the PF1 facility was in full operation, a strategic plan to transition multi-use operations to single use was implemented, with the final TFF buffer exchange step being one of the first to make the switch. The approach for transitioning was simple and straightforward. First, vendor descriptions were used to identify a TFF system which would best meet the needs of a CMO, and then performance was evaluated in three stages: 1) demonstrating suitability in scale-down models, 2) scale-up to a PF1 manufacturing process, and 3) assessing reliability over multiple runs with multiple lots of TFF filters.

FILTER SELECTION

For a CMO which manufactures a wide range of biomolecules, key attributes for choosing a single-use TFF filter system were suitability for cGMP manufacturing, cost, type of filter membrane, a large product line for both pore size and filter area, and an option for a completely single-use flow-path. The Sius single use TFF system from Novasep addressed all of these points and was thus chosen for experimental evaluation. These single-use cGMP TFF cassettes cost approximately one-fifth of typical re-usable TFF cassettes, but use the same type of membrane chemistry (modified polyethersulfone [PES]) common to many other filter applications and have a favorable flux performance when compared with traditional multi-use filters (3). In addition to the standard mPES membrane (Prostream), a more hydrophilic version of the mPES membrane (Hystream) was also available for challenging applications, such as very high final protein concentrations and particularly hydrophobic proteins. As a CMO with both clinical and large-scale commercial manufacturing capabilities, a notable feature is that the vendor's re-usable TFF cassettes are available with membranes identical to the single-use product (cassette construction methods distinguish the single-use and reusable systems). This complete scalability provides a low-risk means of transitioning to re-usable filters later in a product's lifecycle, if the protein therapeutic were to eventually need large-scale manufacturing where the economics are more favorable for re-usable systems (2). Membrane pore sizes range from 1 kDa MWCO to 0.65 µm, and standard cassette sizes range from 0.01 to 2.5 m2. This complete product offering provides the means of establishing a platform TFF process with built-in flexibility to handle a wide range of client proteins. For the early stage clinical programs typical for the PF1 facility, rapid and economical process development is usually an objective, and simple platform processes are one means of achieving this. Finally, although the single-use TFF cassettes are compatible with traditional cassette holding devices, an optional single-use flow-path insert is also available that provides the same type of sanitary connections. The disposable filter plate insert, made of USP Class VI polypropylene, isolates the process fluid from contact with the clamping surface of the cassette holder, facilitating product changeover and eliminating sanitization of the cassette holder as a process step.

Besides the filter itself, decisions were also required on the other components of the system that would be evaluated. Because product changeover considerations were a key driver for single use TFF in PF1, a system consisting entirely of single-use product-contact surfaces was the ultimate goal, including product containers, retentate reservoir, pump head and/or tubing, and in-line sensors, as well as the TFF cassette and flow-path insert. Such a system is readily achievable with current technology, although for this evaluation, the single-use sensors were omitted from the otherwise completely single-use flow-path. This omission was to avoid the additional complexity in a cGMP setting of requiring metrology to simultaneously transition to new sensor technology. A subsequent stepwise transition to fully single-use product contact surfaces is relatively straightforward, as both single-use sensors and flow-path components use compatible connections. Including additional single-use components does affect the economics of TFF operations to a single-use system. It was previously demonstrated that single-use TFF filters alone (i.e., in a re-usable flow-path) had a cost advantage over re-usable filters for filter areas up to at least 3 m2 (2). This advantage is true for any number of annual production batches, although the cost advantage increased significantly when campaigns were fewer than about five batches. For much larger filter areas, the effect of filter costs becomes more significant than labor, so the single-use cost advantage was specific for short manufacturing campaigns. When adding a single-use flow path into the equation (see Figure 2), overall costs are increased but are still favorable over a re-usable system for the typical clinical campaign of two to five batches, the primary clients of the PF1 facility.

Figure 2: Example of a single-use flow-path. A) Flow-path insert. B) Flow-path insert assembled with tangential flow filtration (TFF) cassettes in nonproduct-contact stainless-steel holder. C) One example of a complete single-use flow path using a bioprocessing bag as a retentate reservoir. Note: PT is single-use pressure transducer, FT is flow transmitter, TT is temperature transmitter, Cd is conductivity sensor. D. Cost savings of single-use TFF relative to traditional multi-use TFF as a function of total number of production batches (i.e., the number of times a multi-use filter would be used) and the degree to which single-use technology is incorporated into the TFF system. Cassettes only: Sius single-use cassette as a straight replacement for a re-useable cassette with a standard flow-path. Cassette + PFI: Sius filter cassette and single-use flow-path insert together as a replacement for a standard filter. Complete flowpath: Single-use components for all product contact surfaces in the entire system used to perform TFF. Assumptions for single-use cost savings: filter costs are 80% lower, labor is 60% lower, and buffer/water for injection usage is 75% lower. As the Sius filter is provided presanitized, buffer usage assumes that pre/post sanitization is not performed, although for re-useable flow path options, sanitization of certain components may be required and savings for buffer usage may be somewhat less.

IMPLEMENTATION

The approach to new filter implementation at CMC Biologics involved filter evaluation at small-scale in process development followed by scale-up to a full-scale engineering run, then use in cGMP production. After selecting Sius as the candidate single use TFF filter for PF1, a monoclonal antibody (MAb X) was chosen as the initial experimental model (see Figure 3). MAb X had previously been manufactured using a traditional re-useable TFF filter and was known to tolerate both the standard TFF process and final product concentration without issues. It also provided a baseline for performance parameters such as flux and step recovery. However, a detailed comparison between the two filters was not a focus of the evaluation, as switching to single-use technology was a strategic decision. Using vendor-recommended operating parameters, a simple buffer exchange process was performed in the Development laboratories with a bench-scale 0.01 m2 Sius-LS Prostream mPES TFF cassette. Product load was 270 g/m2 , which is typical for many TFF processes, although this was 2.5-fold higher than the original manufacturing process that had used oversized filters for faster operations. Buffer exchange with the Sius filter, as measured by conductivity, matched predictions for a zero retention coefficient, the expected result for low-molecular-weight buffer components using a 30 kDa MWCO membrane. Product yield was high at 96%, which was equivalent to the original manufacturing process. Product quality attributes, such as percentage of aggregates, were also unaffected by the TFF operation using either filter. Flux was slightly higher with the Sius, averaging 53 L/m2 /h (LMH) compared with 46 LMH with the re-usable filter in manufacturing, despite the higher protein load and lower transmembrane pressure (TMP).

Figure 3: Evaluation with MAb X. A) Process comparison. The re-useable tangential flow filter from the original PF1 manufacturing process and laboratory-scale and PF1 manufacturing-scale Sius filters were all 30 KDa molecular weight cut-off and used cross-flow rates of 360 L/m 2/h. Flux for the concentration and diafiltration stages of the process are shown in the upper graph, and operating parameters in the lower table. B) Buffer exchange for laboratory-scale Sius. Conductivity measurements at various points in diafiltration are plotted against the theoretical conductivity assuming 0% retention of buffer components. Predicted % residual buffer is also shown.

Mab X was then used to address the second objective of process scale-up to PF1 for a 500-L production-scale engineering run. Product was loaded at 210 g/m2 , somewhat less than in the lab-scale trial but close to twice the load of the original process with the re-useable membrane. Because the single-use system eliminates several pre- and post-use operational steps (see Figure 1), this time savings more than compensates for longer processing times during UF/DF from the larger load. Both flux and yield for the single-use filter were comparable between lab-scale and GMP manufacturing. Likewise, product quality was consistent. Operationally, no issues were encountered implementing the system in cGMP manufacturing, and the design was sufficiently similar to traditional multi-use TFF that little additional operator training was required.

Sius TFF cassettes were next evaluated with two problematic proteins to determine whether the system might provide a platform buffer exchange process for the wide variety of client projects performed at CMC Biologics (see Figure 4). Protein Y was a large multimeric protein that was prone to aggregation, and it was evaluated using the same membrane chemistry as had been used for MAb X (30 kDa molecular weight cut-off, Prostream). Again, the filter was first tested at small-scale in development followed by a full-scale engineering run before use in cGMP manufacturing. Performance was similar at both scales. To minimize risk of aggregation, the final target concentration in the TFF step was only slightly higher than the target for final drug substance. Thus, very little buffer volume could be used for a final system flush, providing a challenge for product recovery. TFF performance was good simply using vendor-recommended conditions, with essentially quantitative recovery in PF1 cGMP manufacturing. More importantly, no increase in aggregation was observed as a result of the operation. Protein Z was a fusion protein for which a final target concentration of 50 mg/mL was evaluated in the development group. For this application, the more hydrophilic Hystream mPES membrane at 50 kDa MWCO was used, as it is the vendor-recommended filter for high protein concentration applications. Only minor process optimization was required to achieve an average flux of 96 LMH during diafiltration for rapid processing time, which included performing the diafiltration step at 20 mg/mL protein concentration at an increased cross-flow rate of 900 LMH before a final concentration step to 50 mg/mL. Product quality was maintained through buffer exchange. This high concentration process was not scaled up because of a change in project requirements.

Figure 4: Evaluation with Protein Y and Protein Z. A) Tangential flow filtration (TFF) of protein Y at manufacturing scale in PF1. The table on the left shows process parameters, flux during concentration and diafiltration are in the graph on the right. B) TFF of protein Z in a small-scale experiment. The table on the right shows process parameters, flux and cross flow rates during concentration and diafiltration are in the graph on the left.

The final stage of evaluation was to assess full-scale process consistency in cGMP manufacturing. Six batches of MAb X and five of Protein Y were produced using the Sius TFF system for final buffer exchange (see Table I). Data were obtained from routine cGMP operations as real-world examples. As such, flux and TMP are simply averages of data points periodically recorded manually in manufacturing batch records. One objective of this evaluation was to determine whether lot-to-lot variation in filter cassettes might be an issue for the single-use system. A total of four different lots of Sius filters were used over the course of the MAb X campaign and two lots for the Protein Y campaign. Product yield was consistently high across all manufacturing batches and processing times for UF/DF were rapid and predictable, within ranges expected for manually performed operations.

Table I: Process performance of single-use buffer exchange step in cGMP manufacturing

CONCLUSIONS

Transitioning from multi-use to single-use TFF was a simple and straightforward process and did not require a significant investment of either time or resources. The transition was facilitated by a stepwise approach to a fully single-use flow path by considering a change to single-use sensors in cGMP manufacturing as an independent project. The Sius single-use TFF system is well suited for a 500-L production scale CMO manufacturing facility, or for a typical pilot plant. Performance was comparable or slightly better than the traditional re-usable filters that had previously been used, and implementation in cGMP manufacturing was straightforward. Changes to batch records and operator training were essentially limited to eliminating several pre-and post-use operations. With a fully disposable flow-path, the system greatly simplifies product changeover, reduces overall operating time by reducing the number of process steps, and the economics are still favorable relative to multi-use filters for the typical clinical-stage manufacturing campaign.

BARBARA A. THORNE was a visiting scientist, STEVE WAUGH* is director, downstream process development, TODD WILKIE is a process technology specialist, and JOE DUNN is a principal development scientist, all at CMC Biologics, Bothwell, WA. MICHAEL LABRECK is global product manager, TangenX TFF Products at Novasep, *swaugh@cmcbio.com. B. A. THORNE is currently at Celladon Corp., San Diego, CA.

REFERENCES

1. W.G. Whitford, BioProcess Intl. 8 (11), 34–42 (2010).

2. M. LaBreck and M. Perreault, "Guide to Disposables Implementation," supplement to BioPharm Intl. 23 s32–s38 (2010).

3. E. Miller-Cary, G. Annathur, and J. Glynn, presentation at IBC's 6th International Single-Use Applications for Biopharmaceutical Manufacturing Conference (La Jolla, CA, June, 2009).