Automating the process for commercialization
The process for generating this autologous DC immunotherapy has been consistent and robust for the RCC and HIV indications
during Phase II clinical trials. Elutriation and culture bags have moved key cellular processes to functionally closed, single-use
disposable units. However, some manipulations and processing are still performed using manual methods, including open manipulations
in biological safety cabinets. Although this approach may be feasible for the number of batches required for clinical trials,
it is not practical for commercial manufacturing because every patient requires a new batch of material to be produced.
Advanced RCC has a relatively low incidence of an estimated 9400 new cases in the US in 2010 (11–13). This number alone would
mean a considerable number of batches to manufacture per year. An HIV product would likely require manufacturing for more
than three times as many patients per year in the US, compared with an advanced RCC product. Even more staggering is the number
of patients for Provenge (sipuleucel-T), the first approved autologous cellular therapy produced by Dendreon. Provenge was
approved for metastatic castrate-resistant (i.e., hormone refractory) prostate cancer, an indication for which more than 100,000
patients are treated per year (11, 14). These numbers of batches per year require feasible processing methods to meet the
demands of commercial manufacturing for autologous cellular therapies.
If a company used the method described above to manufacture an autologous DC immunotherapy, which could generate years of
drug product with one leukapheresis, the process scale would remain the same for commercialization. The company would not
need to scale up for autologous therapies. The question would be how to scale out or address the throughput needed for commercialization.
Answering this question was the goal of an automation project that developed novel manufacturing equipment. The cornerstone
of the developed approach is the incorporation of single-use, functionally closed disposables throughout the process. This
method was recognized as crucial for autologous cellular-therapy manufacturing to eliminate cross-contamination concerns and
minimize turnaround time between processes, given the throughput needs.
Automated cellular equipment.
Personnel began the process by adapting the cellular processing methods to incorporate elutriation and culture bags. Equipment
to perform the remaining cellular-processing steps at the scale required for autologous cellular therapies, however, did not
exist. Therefore, two similar instruments were designed with the functionality to perform each of these process stages, including
processing the autologous plasma collected during the leukapheresis procedure for use in drug-product formulation, and designing
custom disposable sets to handle the nuances of each process. The reason for designing two instruments was to create one for
plasma and monocyte processing, which are less complex. The second instrument addressed the unique needs and volumes required
for mature DCs, such as the electroporation step, including handling the addition of precise volumes of RNA normalized to
the DC concentrations, and formulation.
The programming and disposables were designed so that reagents and the cellular inputs for each process were connected to
the appropriate disposable set using standard functionally closed disposable manipulation methods (e.g., tube welding). The
design incorporated the removal of culture bags or other process outputs using tube-sealing methods. Therefore, the cellular
equipment would never be exposed to patient material, and all products and processing will be closed, thus eliminating potential
contamination events and product losses resulting from open manipulations. Although it is always crucial to minimize batch
failures, the need is greater for autologous cellular therapies, given that each batch is specifically manufactured for a
patient, and the patient undergoes leukapheresis to provide the starting cellular material for manufacturing. Because the
high variability of the biological starting material influences manufacturing success, personnel need to incorporate any possible
additional controls during manufacturing to ensure the generation of product. Processing with single-use, functionally closed
disposables and automated methods is a way to significantly improve process control.
The development of the automated cellular equipment and associated disposable sets began with identifying the key processing
steps and needs. Moving from elutriation to culture (or freezing to thawing and then culture) required media-exchange steps
and the ability to resuspend and distribute cells in a functionally closed disposable component. Similarly, harvesting the
DCs, electroporation, culturing following electroporation, and final formulation all required cell-concentration and media-exchange
steps. Methods for reliably and accurately addressing this manipulation of cells were developed for even the small-volume
manipulations required for electroporation or formulation (i.e., 5–30 mL) while not compromising processing time when large-volume
manipulations (large volume for an autologous cellular therapy means as much as 5 L) are required. Overall, the time required
for cellular processing using automated equipment is similar to that for the manual methods. The majority of the time required
for cellular processing, however, is dedicated to incubation to generate DCs from monocytes, followed by culturing for maturation
and culturing for recovery after electroporation.
The biggest challenges to resolve were the management of losses and the maximization of recovery at each step. Minimizing
the manipulations for harvesting procedures and media exchanges was critical to ensure that process efficiencies (which were
measured by the percentage of monocytes in the initial leukapheresis resulting in RNA-electroporated, mature DCs vialed as
drug product) for the automated process were similar to those of manual processing.
Table II: Results for the four initial feasibility runs using the developed automation equipment and functionally closed disposables.
Four initial cellular-feasibility runs performed on prototype automated equipment using the developed disposable sets demonstrated
the cellular equipment's ability to perform the processing efficiently with well-controlled, small volumes (see Table II).
The formulation of the drug product was on target; it had high DC viability post-thaw. The number of doses produced met expectations;
the variability in dose numbers related to the variability in the number of starting monocytes present in the leukapheresis
for each run. The drug-product immunophenotyping results for these automated runs confirmed identity and consistency in quality
(see Figure 3). Results were similar to those generated in clinical manufacturing (see Figure 2).
Figure 3: Post-thaw immunophenotyping results confirmed the identity and quality of the dendritic-cell products generated
in the four initial feasibility runs using the developed automation equipment and functionally closed disposables. CD is cluster
of differentiation, and HLA is human leukocyte antigen.
Automated RNA equipment.
As the cellular automated equipment and disposable sets were developed, equipment also was developed to amplify autologous
RNA from a tumor sample. Though various platforms for automated equipment for nucleic acid manipulations exist, they are generally
based on high-throughput methods and open manipulations of plates. For isolating and amplifying nucleic acids for an autologous
therapy, this type of equipment could potentially be placed in a barrier isolator to achieve the isolation required for manufacturing.
The cleaning requirements between patient samples to prevent cross-contamination, however, would be time consuming. Ensuring
that existing automation platforms could withstand vaporous hydrogen-peroxide decontamination between processes would have
required additional instrument development. Also, the cleaning validation would have been extremely challenging because the
products generated were nucleic acids. The concept, therefore, was to use a functionally closed disposable container for processing,
and to design that disposable so that patient material was never in direct contact with the equipment (see Figure 4).
Figure 4: Prototype equipment for automated autologous RNA processing.
The developed RNA disposable container had two main components. The first component was a rigid tray that incorporated all
that was necessary to isolate and process the nucleic acid (e.g., pipette tips, reagents, mechanism for nucleic acid isolations
and purifications, and PCR plate for all incubation steps). The disposable container also includes spectrophotometer cuvettes
and specially designed volumetric cuvettes required to determine the concentration and volume of the isolated and amplified
nucleic acids. These cuvettes ensured that the equipment can calculate yields and the volumes required for concentration normalization.
They also enabled the equipment to perform the entire process without interruption or data from an outside source.
The second component was a flexible barrier with an incorporated pipette head. This flexible barrier was sealed onto the rigid
tray to generate the closed RNA disposable. When closed, the flexible barrier enabled the six-axis robot arm incorporated
in the equipment to access all areas in the rigid tray to perform the liquid transfers and other manipulations required for
processing. Along with the robotic arm, the automated RNA equipment incorporated the thermal cycler needed for PCR and all
incubation steps, as well as the spectrophotometer needed to determine concentration. In initial feasibility runs, the prototype
automated equipment and functionally closed disposable container generated amplified RNA comparable to the amplified RNA generated
using current manual clinical processing methods, thus demonstrating that this automated concept is appropriate for manufacturing
drugs for oncology indications. This concept can be readily adapted to infectious-disease indications to amplify RNA from
a viral sample.