 E-mail
for more information
Company's
other products
E-mail
to a colleague
See
similar products
Printer
friendly format
|
|
Caprylate Chromatography
A "clean-slate" approach to removing harmful viruses
Jonathan Kent, Ph.D.
Chromatographic separation techniques have been used for decades in the isolation and purification of pharmaceutical and biopharmaceutical products. In the process to separate therapeutic human proteins from donated plasma—for example, immunoglobulin for intravenous injection (IGIV)—steps have been added to the process to help ensure the removal of potentially harmful viruses such as HIV, hepatitis C, and more recently, infectious proteins called prions.
The original purification process used multiple precipitation steps that resulted in the IgG protein being taken out of solution and resolubilized repeatedly. This method, while effective, was also potentially damaging to the protein. These repeated precipitation steps can potentially denature proteins and create aggregates that may render a portion of the final product ineffective. Later, the addition of a solvent-detergent step to the process helped achieve viral clearance, but also resulted in yield losses.
For the production of IGIV, a biological therapeutic essential to the survival of a variety of immunocompromised patients, Talecris Biotherapeutics scientists set out to recreate the process used to separate IgG from fractionated blood plasma. They worked diligently to create a process that would more efficiently isolate IgG while integrating the critical viral removal and inactivation steps necessary to help ensure the safety of this life-enhancing product. The solution would have to reduce the steps and time involved, provide for a final product with exceptional consistency, and increase extraction of IgG from donated human plasma, the raw material for IGIV manufacture that is in constant short supply even as the need for this life-enhancing therapy for patients with primary immune deficiencies goes on unabated.
Talecris Biotherapeutics’ award-winning manufacturing site in Clayton, NC, where Gamunex is made. |
Process development scientists at Talecris were able to successfully and efficiently achieve the goal of isolation and purification while integrating effective viral removal steps into the unique IGIV process used to manufacture Gamunex (Immune Globulin Intravenous (Human) 10%). This new patented purification process, combining caprylate and ion-exchange chromatography, has proven to be far more efficient than the method first pioneered in the 1950s.
Tiny solution to a major challenge
Talecris scientists turned to a naturally occurring salt of octanoic acid, called caprylate. We were not unfamiliar with this tiny chemical. Caprylate has been used for 50 years to stabilize albumin and it was known, at the right pH, to be a very efficient viral inactivation agent. However, even we were somewhat surprised at the 8-carbon fatty acid’s added ability to streamline the precipitation and chromatographic process of IGIV.
In the 1990s, the solvent detergent method was developed for viral inactivation. Detergent disrupts the envelope of certain viruses and is a simple means to target a variety of viral pathogens. However, the detergent had to be removed in the final process. With oppositely charged ends of the chain molecule, caprylate acts like a detergent by binding to and destroying viral envelopes. Using caprylate, a natural, plant-derived chemical, in the process for purification of IgG seemed logical, especially if it would allow us to precipitate unwanted proteins while leaving IgG soluble. When our scientists began looking into the dual role of caprylate to both selectively precipitate proteins and inactivate viruses, several papers had already been published that described the use of caprylate to precipitate proteins.
In the previously used method, the immunoglobulin paste feedstock underwent multiple precipitation steps. Each time the IgG molecule was separated by this means, it had to be resuspended in solution, which took a toll on the delicate molecule.
Caprylate was found to be an ideal way to effectively remove many impurity proteins as precipitate, while keeping the immunoglobulin soluble during this entire process, thus minimizing potential damage to the IgG molecule. In fact, under very precisely defined conditions, caprylate is very effective in precipitating non-immunoglobulin proteins along with enveloped and non-enveloped viruses. The beauty of caprylate is the simplicity with which it can help integrate the removal of both unwanted proteins and viruses, while keeping IgG soluble.
The process defines the product
In the isolation and production of complex biologics, the process defines the final product. With this in mind, Talecris committed to an entirely new facility to produce Gamunex rather than retrofitting an existing facility. Completed in 2003, our Clayton, NC facility for producing Gamunex runs 24 hours per day, 365 days a year. The facility is comprised of two identically configured processing trains, which adds flexibility and increases processing throughput.
The automated facility was designed with two processing “zones,” using the concept of areas of separation based on viral clearance. Zone 1 is where the paste dissolution and caprylate precipitation part of the process takes place. This process occurs in two steps: the first is a viral-removal step, while the second is a viral-inactivation step. These steps are called MIC-1 and MIC-2, for Material in Caprylate Steps 1 and 2. The caprylate is the agent that precipitates either the protein or the virus in MIC-1, which gets caught by the depth filter. The filtrate—with the target IgG molecule in solution—will pass through the depth filter while remaining in solution. MIC-2 involves an incubation step at a slightly elevated temperature. This caprylate incubation step is dedicated to the inactivation of enveloped virus. Throughout the entire Zone 1 process, IgG remains in solution, minimizing stress on the protein.
Once the Zone 1 processing is complete, the process solution moves to Zone 2 where it is subject to ion exchange chromatography. Two charged resins are used in two different, fixed-bed chromatography columns. The first column is 1.8 m in diameter and 20 cm high, while the second column is 1.2 m in diameter and 25 cm high.
The first column contains a strong anion exchange resin, while the second column contains a weak anion exchange resin. These columns are loaded in flow-through mode, which allows the desired, unbound IgG to flow through both columns. This critical polishing step is run under very carefully designed, well-controlled conditions that ensure that undesired proteins and caprylate remain bound to the columns while IgG is allowed to pass through.
During research and development, a narrower column than is currently used was tested. It was found that caprylate bound so tightly to the anion resin, that a thick layer of caprylate at the very top of the column would actually restrict the flow through the resin. One end of the caprylate molecule is non-polar, so every positive charge on the anion resin would essentially get caprylate bound to it. This was actually a good problem to have and was quickly solved by using wider columns.
Following anion exchange chromatography, the protein solution is at a relatively low concentration of about 0.5%. The next step in the process is ultrafiltration/diafiltration (UF/DF). By pushing the solution through a set of ultrafilters with a specific molecular weight cutoff, the concentration of IgG is initially increased about 10 fold. Subsequent diafiltration allows for the protein to be “washed” by replacing small solutes with water molecules. The final steps include final concentration and bulk formulation to 10% IGIV-C liquid.
These initial sterile bulks are combined from multiple trains and filtered into a sterile filling bulk that is then sent to the adjacent sterile filling facility (SFF) in Clayton. After the vials are filled, they are incubated at room temperature for 21 days at about 25 C. This final incubation is a dedicated viral inactivation step. At this temperature and under low pH, our Pathogen Safety group has demonstrated a highly effective final viral inactivation step.
Producing Gamunex using this unique caprylate process, combined with chromatography, provides quite an efficient process for producing this life-saving therapy for the patients who depend on it.
Efficiency and consistency
The entire process takes between 30 and 35 hours, from the time you initiate a batch with Fraction II + III paste to the time you have an initial sterile bulk. The previous solvent-detergent-based process took anywhere from 100 to 130 hours. The process consistently yields 25 to 30% more IgG per unit of starting II + III paste when compared to the old solvent-detergent-based process.
The new process minimizes the harsh steps. Two ethanol precipitation steps used in the previous method have been replaced with caprylate precipitation, which allows the IgG molecule to remain in solution throughout the entire process. Detergent is no longer added, and therefore its removal is no longer required. The final product is formulated at a lower pH, which enables additional viral inactivation. From the time of II + III paste dissolution to filling, the immunoglobulin never leaves the liquid phase, which dramatically reduces the possibility of protein denaturation. The overall consistency and efficiency of our process has clearly been demonstrated, year after year, batch after batch.
The facility producing Gamunex is operated by a highly trained staff consisting of four production teams who work rotating 12-hour shifts. Each team consists of 23 people, including a shift supervisor. Although the facility is highly automated, the care taken by each member of the production team is critical to the success of this high-throughput manufacturing facility. Highly experienced engineers, instrumentation and maintenance technicians, and technical support staffs in Operations and Quality work diligently and conscientiously to help ensure that each batch of Gamunex produced is of the highest possible quality for the patients whose lives depend on it.
Jonathan Kent, Ph.D. is the Technical Support Manager for Gamunex at Talecris Biotherapeutics, Inc. He may be contacted at ChromatographyTechniques@advantagemedia.com.
Biomatrica
5627 Oberlin Drive, Suite 124
San Diego, CA, 92121
|
