Production of the tumor antigens is the second step in the process. Manufacturing considerations at this stage of the process include whether the cells: 1) express the TAA of interest, 2) yield enough TAA to manufacture vaccine, 3) can be used to make a desired stable transformed line, and 4) can be grown under acceptable cGMP cell culture conditions. The cell culture growth conditions must be set in regard to the type of culture vessel to be used (e.g., flasks, "cell factories," closed bags, or roller bottles), whether the cells can be adapted to serum-free media or whether traceable animal products are required in the growth media, and the qualification of the manufacturing site for expanding the tumor cell lines and preparing the TAAs.

Manufacturing facility qualification is less of an issue for allogeneic tumor cell lines than for autologous tumor cells, since allogeneic tumor cell banks can be established and a single cGMP manufacturing site can be qualified for production of TAAs to meet commercial demand. In contrast, the use of autologous tumor cells presents several unique problems in maintaining manufacturing control and product consistency. Since the tumor cells are obtained from individual patients, the first decision is whether to prepare the TAAs at the collection site under a qualified procedure or to ship the cells to a central location where the TAAs can be prepared in a validated cGMP manufacturing facility. Regardless of where the vaccine is manufactured, establishing product consistency between TAA products will be difficult when using unique autologous tumor cell isolates.

The final step in the process is QC release of the formulated and filled TAA vaccine product for patient use or drug substance for loading into DC. The following issues should be considered at this step. First, cells maintained in culture for extended periods can become contaminated with mycoplasma, thus, precautions and testing are required to ensure the TAAs are free of mycoplasma. Second, when killed tumor cells are used as the vaccine, the process should be validated to ensure that the tumor cells are not capable of progressive growth in vivo. Third, a decision is needed as to whether dosing will be based on TAA amount or cell number. Finally, TAA lysate and killed tumor cells can be filled into vials and frozen for storage prior to injection, assuming that stability-indicating assays are in place and that the desired stability can be established for the product.

The initial steps in the manufacture of a DC vaccine involve enrichment of the precursor cells from the patient's apheresis blood product. Once isolated, the DC precursor cells are placed in cell culture with the appropriate mixture of cytokines and loaded with TAAs. Upon completion of the cell culture step, the DC are harvested, formulated, and filled for injection into the patient. The vaccine can be given to the patient as either freshly prepared DC or from a batch of frozen DC.

Since the DC are derived from the patient, processed ex vivo, and formulated for injection, the same issues outlined above for the use of autologous tumor cells for the manufacture of TAAs apply here to the preparation of autologous DC vaccines. These include: how to qualify a cGMP facility(ies) to manufacture vaccines with cells collected at various locations; how to validate manufacturing controls for a vaccine product based on unique blood cell populations; and how to establish vaccine consistency for a product derived from individual patients. Whether the DC vaccine needs to be made fresh or can be frozen requires considerable characterization of the product and correlation with clinical efficacy data.

Quality Control and Release Testing

Cancer vaccines, whether TAA- or DC- based, must be released to the same criteria as those required for other pharmaceuticals, i.e., sterility, identity, purity, and potency. Sterility testing must establish that these products are negative for gram and Hoechst stains, and there is no outgrowth of mycoplasma and bacteria at 14 days and fungus at 30 days. Since expediency is key to the treatment of cancer patients, initial vaccine dosing may begin before sterility testing is completed. In those instances, the vaccine may be released with a minimum amount of sterility testing completed, usually a gram stain, Hoechst stain, and PCR testing for mycoplasma. The patient must be monitored for symptoms of infectious diseases and appropriate treatment initiated if necessary. Therefore, validating a sterile process early in the development of these products is advisable.