Vendor Notes: The Biovest AutovaxID: Enabling the Promise of Personalized Medicine

ABSTRACT

The AutovaxID is a self-contained, completely enclosed, fully automated hollow fiber bioreactor that permits rapid, efficient scale up of patient-derived cells. It is based on hollow fiber bioreactor technology and is an ideal system for high-density cell culture and the production of monoclonal antibodies or other therapeutic proteins.

With the era of personalized medicine upon us, developers of novel, patient-derived cell- and vaccine-based therapies face two enormous challenges: generating safe products under good manufacturing practices (GMP), and delivering these new treatments at reasonable cost.

Platform technologies can standardize and streamline manufacturing operations for personalized treatments. Influenza vaccine, for example, is developed and manufactured rapidly through well-known platform techniques involving egg-based virus culture. For treatments derived from a patient's own cells, an ideal cell culture platform approach should be:

• Compact, to minimize the size of manufacturing facilities (especially for treating diseases with large patient populations)

• Rapid, to permit treatment of acute conditions

• Composed of disposable elements for all product contact components, to maximize speed and manufacturing flexibility

• Closed and self-contained to improve regulatory compliance.

• Automated, to enhance productivity and reduce operator induced errors

In 2001, Biovest International began investigating options for manufacturing patient-derived therapies in a compact, production-worthy cell culture device that required significantly less human interaction than traditional stainless steel or multiple flask-based cell cultures.

The design was driven by ongoing development of a vaccine against non-Hodgkin's lymphoma, which is currently in Phase 3 testing at several sites in the United States and Russia. The challenge was to develop a scalable, commercializable platform technology, or an instrument that could run in parallel with multiple other devices. Moreover, this production methodology needed to be suitable for production in a cGMP facility in which multiple, patient-derived products are manufactured simultaneously.

Biovest scientists recognized early on that no such personalized biologic could be commercialized without a significant reduction in process complexity compared with conventional cell culture. Biovest believed that regulatory agencies would support a device that could eliminate cross-contamination and minimize operator errors.

NECESSITY, THE MOTHER OF INVENTION

In the mid-1980s, through its predecessor company, Cellex Biosciences, Biovest was the first vendor to offer commercial-scale hollow fiber bioreactor systems. These bioreactor systems included Acusyst Technology (mini-Maximizer, Maximize, Xcell, and Xcellerator), which provided hollow fiber technology suitable for research to commercial scale.

In 1999, the National Cancer Institute granted an award to Biovest to evaluate patient-specific cell line–derived antibodies to treat follicular lymphoma, and subsequently became the commercial sponsor of this cancer vaccine. As part of this cooperative research and development agreement (CRADA), Biovest began designing a novel production device that could be used in a multiproduct facility to facilitate the manufacture of patient-specific products as per US Food and Drug Administration GMP guidelines.

Development of the device was slow until Accentia Biopharmaceuticals became a majority shareholder of Biovest. Accentia recognized the value in emerging personalized medicines and believed this patient-specific treatment showed tremendous promise in improving and enhancing the quality of life. After acquiring Biovest and its scientific assets in June 2003, Accentia immediately invested in the lymphoma vaccine (BiovaxID) and the platform technology (AutovaxID) required for ultimate commercialization. BiovaxID, which confers immunity against non-Hodgkin's lymphoma, is currently in Phase 3 clinical trials.

As our personalized lymphoma vaccine product(s) progressed through clinical testing, the deficiencies of conventional cell culture manufacturing became evident, promoting a complete rethinking of the production process. It became clear that the process ideally required a device that could offer sample containment or segregation, high-throughput, a small-footprint, and the ability to concurrently manufacture thousands of patient-specific products in one facility. Based on this premise, the AutovaxID emerged after several years of development effort that combined cell culture, engineering, electrical, and mechanical expertise.

The AutovaxID is a self-contained, completely enclosed, fully automated hollow fiber bioreactor that permits rapid, efficient scale up of patient-derived cells. Because it is based on hollow fiber bioreactor technology, the AutovaxID device is an ideal system for high-density cell culture and production of monoclonal antibodies or other therapeutic proteins. A commercial version of the AutovaxID became available in October 2006.

CAPABILITIES

AutovaxID is well suited for growing any antibody-secreting cell line, including hybridomas and Chinese hamster ovary (CHO) lines. In the case of our lead product, BiovaxID, patient-specific cell lines were created by fusing patient-derived tumor B-lymphocytes with an established murine heterohybridoma cell line. Since B lymphocytes, by themselves, are somewhat difficult to culture, and are poor ex vivo producers of monoclonal antibodies, fusion with another hybridoma line provides them with enhanced ability to proliferate and secrete their unique immunoglobulin. This immunoglobulin (IgG or IgM) is unique to each clonal cell population, and in this case, represents a unique tumor antigen. More specifically, the idiotype, or CD3 region of these immunoglobulins, is unique to each patient. As such, fusing cancerous B cells with an established hybridoma line gives us the ability to manufacture large quantities of tumor-specific protein, which is purified and used to immunize the patient against recurrence of lymphoma. In Phase 2 studies, BiovaxID has shown that after nine years 95% of treated patients are still alive and 45% remain disease free.

In addition to BiovaxID production, the AutovaxID is used routinely at the Biovest National Cell Culture Center to produce monoclonal antibodies for academic investigators. We believe the AutovaxID holds great promise in research settings because of the instrument's ease of use, robust operation, and high productivity.

Figure 1

A good deal of the AutovaxID's ease of use derives from its facile, straightforward setup. Once cells are introduced into the AutovaxID, the instrument takes over all significant aspects of the operation. Where preparation and setup can typically take several hours in conventional hollow fiber bioreactor systems, these operational steps have been reduced to less than five minutes in the AutovaxID. By eliminating this complexity, technician error and labor are significantly reduced, especially when multiple instruments are needed for concurrent production of numerous products.

The AutovaxID incorporates several features that lead to enhanced automation of this device. Most notably, the system incorporates real time CO₂ measurement as a means of monitoring and controlling lactic acid concentration in the culture. Based on a user-defined lactate setpoint, media delivery rates are automatically adjusted as the cell population expands. This provides on optimal feed strategy without need for expertise or waste of expensive media. The instrument provides many of the control and process optimization features of bioreactors that are much larger, with the additional advantages of versatility, small footprint, and ease of use.

Biovest's National Cell Culture Center routinely uses the AutovaxID for its monoclonal antibody production services. Antibodies have been produced from client-derived cell lines that include mouse and rat hybridomas, human heterohybridomas, and transfected CHO lines. Production levels vary considerably, depending on the cell-specific productivity and intended duration of each culture. Overall, quantities produced ranged from 0.5 gm to 5.6 gm in 45 days. In general, an average hybridoma that produces 15–ug/mL in T flasks can be expected to produce 1–2 grams of antibody in 30 days from the AutovaxID. More importantly, these cultures required approximately 75% less labor than that typically required for conventional hollow fiber bioreactor production instrumentation (10–15 hours per month, as opposed to 40–45 hours per month). As a result, technician time was more available for additional projects, which significantly increased overall productivity of the laboratory.

LOOKING UNDER THE HOOD

The AutovaxID instrument consists of two components: the control unit and an enclosed, disposable bioreactor (cultureware). The disposable component is completely enclosed in a plexiglass housing that slides into, and snaps in place within the instrument. The series of photographs in Figure 2 demonstrates the ease of use of the AutovaxID. Initially, a completely enclosed, disposable culture module is removed from its packaging (middle left) and placed in the control unit (upper left). Next, the culture parameters are programmed into the instrument (upper right), followed by bar code scan (middle right) for materials tracking and data capture. Following this, cells are introduced into the bioreactor and the automated culture process is initiated (lower right). For the duration of the culture, clarified supernatant is continuously collected and stored in the refrigeration compartment of the AutovaxID (lower middle) until the run is terminated, or product is ready for further processing. With conventional hollow fiber instruments, technicians must first prepare the cultureware for installation into the instrument. This may include loading peristaltic pumps, aseptically inserting an autoclaved pH probe, and manually opening and closing clamps to facilitate a preinoculation fill and flush procedure. The entire preparative process may take several hours and is prone to mistake by an inexperienced technician. All these manual operations have been eliminated with the AutovaxID. Technicians need only snap the cultureware into the device and connect a media feed source. Following an automated fill and flush procedure, inoculation and secondary pH calibration, the instrument's process control then allows for unattended operation for the duration of the culture.

Figure 2

The disposable cultureware for the AutovaxID contains all disposable fluid or product-contact components, including the bioreactor and associated tubing, pH probe, cycling and harvest bags, and a gas exchange cartridge to oxygenate and provide feedback control of pH in these densely packed cultures. Note that although the AutovaxID is described as a "closed" system, users must still introduce cells and change media bags, which is normal for any cell culture device.

The AutovaxID control unit contains a built-in computer, touch screen display, and built-in solid-state refrigerator. The entire system can be monitored and controlled remotely via a web browser. The software was designed to maintain process parameter setpoints for pH, lactic acid, and temperature, and also to monitor pump speeds and calculate media volumes. When media reach a critical low point, for example less than five liters, the technician is alerted to replace or add a new container.

Biomanufacturing has benefited for 20 years from hollow fiber bioreactors, whose principal advantages are their disposability and ability to support a large cell population within a very small space. Hollow fiber bioreactor devices are also continuous, densely-packed cultures that facilitate product harvest at high concentrations. This, combined with online product filtration in the AutovaxID, greatly facilitates or eliminates the first step in the product purification process. In hollow fiber bioreactor systems, it is common to harvest protein continuously at concentrations of several milligrams per milliliter, as opposed to micrograms per milliliter in conventional batch processes. As an added benefit, hollow fiber bioreactors conserve costly, high molecular weight growth factors or serum-based additives by retaining these expensive reagents on the same side of the membrane as the cells.

The AutovaxID was specifically designed for use in multiproduct facilities that must manufacture large numbers of patient-specific, cell-based therapies simultaneously and in a relatively compact space. In designing the AutovaxID to include such features as product segregation capability, automation, and software security we believe that we have addressed the key regulatory requirements relating to production of personalized biologics by the instrument.

THE FUTURE OF PATIENT-DERIVED PERSONALIZED THERAPIES

The challenges for developing a bioreactor that addresses the manufacturing needs of personalized therapeutics are daunting. Any application involving the expansion of specialty or patient-specific cells will be extremely labor-intensive and expensive using traditional cell culture methods. As medical science struggles with the regulatory issues and labor burden of expanding cells in culture for personalized treatments, we believe that the AutovaxID platform technology is the only way to cost effectively manufacture and market these products.

AutovaxID has shown its value and versatility for producing patient-specific secreted proteins for therapeutic use, at a cost that makes potentially life-saving therapies accessible to patients with many serious illnesses. While we expect to continue to serve this important area of personalized medicine, we also expect that many new and promising treatments will arise from cell-based therapies. Biovest is currently exploring ways to optimize the AutovaxID for applications which involve recovery of whole cells grown in the AutovaxID for therapeutic use. At present, our challenge is to develop a process that allows for cost-effective growth and efficient recovery of unique or specialized cell types, including stem cells.

Biovest and its parent company, Accentia Biopharmaceuticals, are committed to the continued development and improvement of the AutovaxID instrument for the production of therapeutic materials. Both companies believe that within the next several years it will become a key enabling technology for the emerging field of cell and cell-derived therapeutics.

Mark Hirschel, PhD, is chief scientific officer at Biovest International, Inc., Minneapolis, MN, 800.325.111 ext.206, mhirschel@biovest.com