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.