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View of cleanroom #1 in new vaccine manufacturing facility at Baylor Institute for Immunology Research.

By Nicolas Taquet
Technical Director of Baylor Institute for Immunology Research Technical Director of ODC Therapy, Inc.

Melanoma is a form of skin cancer. The American Cancer Society estimates that there will be 59,580 new cases of melanoma and 7,770 deaths from melanoma in 20051. Since 1999, Baylor Institute for Immunology Research in Dallas, TX has carried out four ‘Investigational New Drug’ clinical trials. These FDA-approved trials have led to a potential cell therapy for melanoma patients.

Development Of A Frozen Vaccine Against Melanoma Skin Cancer

Patients with cancer cannot eliminate cancer cells. The immune cells that protect them from for example, viruses, have become tolerant to the cancer cells. Our strategy is to educate the patient’s immune cells to break this tolerance and direct them to eliminate the cancer cells. To do so, we use the patient’s own dendritic cells (white blood cells that recognize and bind foreign substances) loaded with killed melanoma tumor cell fragments. Patients receive several injections containing their own cultured and loaded dendritic cells. Our research has shown that this treatment is safe and with little toxicity.

This ability to provide a patient-specific cancer treatment has taken years to develop. At first, we were using freshly prepared vaccines which took up to nine days of preparation followed by heavy release testing. The cost of such an approach was very high and it required numerous cell processing centers located very close to the patient’s clinic to provide a fresh, efficient product. We have slowly decreased our manufacturing time by implementing new types of vaccine preparation protocols. Today we manufacture our vaccines in three days. This allows us to produce more vaccines using the same structure and the same number of personnel. We have also developed a frozen vaccine enabling us to ship it anywhere to any patient’s physician. We now release only one batch of vaccine once for each patient. This simplifies and streamlines the process while reducing production costs.

A New Approach To Reach Industrial Production Scale

Today, most biotech companies or academic institutions involved in cell therapies are packaging their vaccines in special cryobags. Because the final product has a small volume, it requires very small bags which are difficult to handle. Filling is also a problem due to complicated tubing systems which can create dead volumes, resulting in the possibility of wasted product – this is critical because every drop of product that we lose will be one less dose for the patient. The vaccine cell concentration is usually very high to help survive the freezing process. Because of the use of a cryopreservant to prevent cell membrane breakage during freezing and the nitrogen-vapor long term storage, many centers are washing the cells prior to injecting them. This step is also complicated when cryobags are used.

Since 1999, Baylor has used plastic screw caps and vials to freeze and store their cells. This process was performed manually within a Class 10,000 (ISO 7) cleanroom inside of a Class 100 (ISO 5) biosafety cabinet. Recently, an M&O Perry P1540 automated filling machine was designed and built under the very strict current good manufacturing practices’ for pharmaceutical quality standards, which has helped in standardizing our vaccine filling. With limited human intervention to prepare the machine at the beginning, the preparation of roughly fifty vaccine doses for one patient takes less than two minutes.

View of filling, sealing, and packaging room showing back of M&O Perry P1540 filling machine and two customs made Accuseal 540 Plus machines.

Custom Ordering Of Cleaned, Stoppered, Sterilized, And Certified Vials

In order to maximize the quality, every component involved in the process is outsourced to specialized vendors. The reagents and ancillary materials come triple packaged, sterilized, and certified. Our quality team inspects each received lot, review the attached certificates and releases them prior to entering the manufacturing area.

The P1540 liquid filler handles pre-sterilized stoppered vials. For our vaccines we collaborated with VWR, West Pharmaceutical Services (WPS), and Wheaton Science Products. Special low-temperature-resistant stoppers coated with West FluroTec from WPS prevents the cryopreservant from possibly altering the stopper (chemo-compatible). The automated filling is made with no puncture of the stopper to keep the West FluroTec film barrier perfect. The stoppers have a septum through which the patient’s physician or a nurse can withdraw the thawed vaccine at the place and the time of each injection. This is the only time the West FluroTec film is breached. The stoppers are cleaned, packaged, sterilized and tested by Wheaton Science products. The 2 ml borosilicate glass vials from Wheaton Science Products are cleaned, packaged, sterilized and tested by them as well. Wheaton then assembles both components and triple packages them under aseptic processing inside a cleanroom.

Outsourcing this critical step eliminates the need of validating in-house the sterilization for a small scale run. Annual audits of our vendors provide external controls and validation. Of the three packaging layers protecting the stopped vials, one is removed when entering the release storage zone. The second layer is removed prior to entering the pass-through leading to the manufacturing area. Finally, the last layer is removed during loading of the P1540’s Class 100 enclosure. An in-feed rotary table allows the vials to reach the conveyor system and different process stations. The stopper is removed from the vial. Vaccine is filled into the vial using a diving needle mechanism. The needle has a specific angle to dispense the liquid on the side of the vial avoiding foaming. The stopper, held by an indexed rotating arm, is then re-inserted into the same vial from which it was removed. The stoppered vial then passes to the capping side of the machine where an aluminum seal (pre-cleaned, sterilized, and tested by Wheaton) is applied and closed. Vials then exit the machine onto a rotary discharge table where two operators can insert them in ultra-low temperature resistant pouches (American Fluoroseal Corporation). Those pouches are sealed using two custom made Accuseal 540 Plus machines. The pouch is the secondary packaging that protects the glass vial from breaking during the various manipulation steps, such as freezing, storage, and vaccine shipment.

View of filling, sealing, and packaging of patient-specific melanoma cancer vaccines.

Development Of Disposable, Single-Use, Filling-Machine Tubing Assembly

The pharmaceutical industry can take the time to prepare a filling machine for a big production, but we have different challenges with our process. Our lots are small (less than fifty doses per patient), with no tolerance for packaging problems, as time is critical once the cryopreservant is added to the vaccine. Therefore, the need for consistent and rapid filling with minimum cleaning of exposed parts between patients is mandatory. Together with Watson Marlow and Value Plastic, Inc., we designed a chemo-compatible, disposable, single-use, filling-machine tubing assembly. American Fluoroseal Corporation assembled, triple packaged, sterilized and tested the filling-machine tubing assemblies. Now we can prepare a lot for one patient and one hour later prepare another one for a second patient with minimal cleaning/change-over in between.

Validation Of Fast, Low-Volume Filling

Because the filling takes less than two minutes in our conditions, the variability between filled vials due to the increased flexibility of the tubing after 20 minutes does not apply. The variability between vials using one tubing assembly to fill fifty vials is low. We verify by weight every filled vial within one lot. Also, the variability from one tubing assembly to another (from one filling of fifty vials to another filling using a different tubing assembly) is expected to be minimal. Therefore, no pump re-calibration should be necessary at the beginning of each new filling. The accuracy of the pump is checked, comparing three pre-defined doses along the filling run.

We are currently collecting data using automated fillings with cultured cells from healthy volunteers to verify the consistency among vials from one tubing assembly to another. Once the minimum variability is determined, we’ll implement the automated filling as our routine. Today, our reference method is manual filling. But on a large scale, to treat the estimated yearly number of stage IV melanoma patients, automated filling is required. Stability data will be collected during our initial Phase II clinical trial to justify this innovative new packaging strategy for a frozen product.

In conclusion, by sharing existing technologies the industry can introduce new and effective therapies. What starts out today as a pilot study can and will have numerous applications tomorrow. It is a very long process for biotech companies and institutions to develop a product, but every standardized resource that becomes available (like standardized automated packaging) can save years in manufacturing.



Pharmaceutical Processing
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