Successful purification of recombinant proteins for use in commercial diagnostics entails efficient and cost-effective use of expensive equipment and components, in close quarters and on tight deadlines, while ensuring and validating cleanliness and purity. n the mid-1980s, Chiron Corp. (Emeryville, CA) entered the commercial diagnostic market with the purification of several recombinant proteins (primarily HIV). Soon afterward, the company formed a diagnostics purification group to produce these proteins and others, including HCV proteins. Today, the Diagnostics Purification Group purifies more than 25 proteins (antigens), licensed and nonlicensed.
Commercial purification of recombinant proteins is an extensive subject. To limit the scope of this article, we focus on three distinct but integral components of a manufacturing operation: process and product segregation, process transfer and scale-up, and in-process testing.
Process and Product Segregation
We may perform a given protein purification process as rarely as once a year or as often as 25 times a year, depending on commercial demand for the protein and on the process's yield. We prefer to perform a process at least once a year to keep the purification staff familiar with the procedures.
A typical process takes one week from cell breakage to final bulk. Yields can range from 20 mg to several grams, depending on the expression level and process scale.
The number of individual processes performed at our facility increased slowly over time, up to a point where we had to start processing two products a week instead of one. Today, we typically perform from two to four processes concurrently. Concurrent processing raises important good manufacturing practices (GMP) issues related to the prevention of contamination and the segregation of processes.
The movement of people, materials, equipment, and waste must be planned to prevent product contamination. Steps must also be taken to effectively segregate processes in the purification area to prevent product-to-product contamination.
Our facility consists of two purification labs and several accessory rooms. Laboratory areas have sealed vinyl floors and sealed ceilings. Air supply is controlled by an HVAC system. Purification areas are pressurized, and while air quality is not classified, environmental monitoring suggests that the air quality does meet Class 100,000 specifications.
The accessory rooms consist of centrifuge labs, cold rooms, and storage rooms. Once a company has established a suitable environment with adequate space for equipment and storage, the next consideration should be process and product segregation.
Careful planning maximizes the use of space and minimizes the opportunity for product contamination. Our purification labs have designated storage and work areas. Chemicals, intermediates, equipment, glassware, and reagents are stored in designated areas, the latter two in dedicated cabinets. Each lab has a reagent preparation area with balances, chemicals, and a pH meter nearby.
Lab benches in rows in the central area of the lab are kept clear of equipment and other supplies when not in use. Lab benches are separated by either an aisle or a divider. Each process is assigned to a specific bench.
The bench area is used for storing components and equipment during protein processing. The primary activity performed in the bench area is chromatography. When the process is finished, personnel clean and store the equipment and clear the area in accordance with written standard operating procedures (SOPs), in preparation for the next process.
The early stages of processing--cell breakage and extractions--are done in small, office-sized centrifuge labs. A centrifuge lab typically houses two to four centrifuges and about 6 ft of benchtop. A typical process starts on Monday and ends on Friday.
All processes have centrifugation steps. The availability of multiple centrifuge labs provides scheduling flexibility and minimizes the likelihood of product cross-contamination.
Processes are segregated spatially. Each handling step (reagent preparation, centrifuging, and chromatography) is performed in a separate designated area.
Another way to separate processes is by the use of dedicated personnel. We assign one or more purification operators to a specific process for its duration, reducing the likelihood of operator error. Process-dedicated personnel are responsible for setting up and performing the process as well as preparing process reagents.
Process reagents have a unique identification system. Each reagent is identified by a part number and a lot number. These numbers are assigned and noted within the body of the batch production record. The lot number of the process reagent is the same as that of the antigen being processed.
We retain samples of all reagents for potential troubleshooting. Once the run is completed and determined to be typical, the retained samples are discarded, as are unused portions. This procedure limits the number of reagents in the labs at any given time and lessens the chance that a reagent may be used in the wrong process.
Another consideration for a multiuse facility is the separation of the manufacturing of licensed and nonlicensed products. If space permits, separating the processing of licensed product from that of nonlicensed product is advisable in order to satisfy regulatory concerns. However, processing licensed and nonlicensed product together is allowable as long as adequate measures are taken to segregate the two.
About two years ago, we annexed an adjacent lab to better handle the increasing demand from Chiron's diagnostic business. We dedicated the original lab to the manufacturing of licensed products, the newly acquired lab to nonlicensed ones.
Previously we had been successfully manufacturing both licensed and nonlicensed proteins in one purification lab, following the segregation measures previously described. All processing, including nonlicensed processing, was performed according to GMPs with valid batch production records.
Process Transfer and Scale-Up
How easily and quickly a process is transferred to manufacturing and scaled up depends on variables such as the antigen's expression level in the starting material, the process methods employed, and the protein's stability. Protein purification processes can vary in complexity from those having simple wash steps and a single chromatography step to those requiring extensive extractions and several chromatography techniques. A typical process design is represented in Figure 1. A new process that necessitates considerable modifications before scale-up or one that requires novel in-process test procedures can slow down completion of the consistency series (production runs used to qualify the process).
Scale-Up Limitations. Some small-scale purification techniques have scale-up limitations. Ideally, development personnel are aware of them, but this is not always the case. Some development personnel have limited exposure to and understanding of the manufacturing environment. For this reason, our purification group established its own process transfer and scale-up group to review and test new processes before manufacturing. This group also serves to troubleshoot problems from any of our established processes.
Certain purification techniques yield good results at a development or small-scale level but may not be scalable. For example, following an ion exchange chromatography step, the product must be concentrated before being passed over a gel filtration column. A process development scientist who needs to concentrate 1 L of ion exchange material down to 100 ml decides to do so using an apparatus that relies on a membrane and nitrogen pressure. When the process design is completed, he or she transfers the process to manufacturing. Manufacturing finds that it needs to scale up the process tenfold to meet demand. However, the nitrogen concentration apparatus has a capacity of only 2 L, so manufacturing must find an alternative method of concentrating the material. The problem with this is that the results (e.g., shearing, foaming, and microbial growth) of using another method are unknown.
Another small-scale technique that is not practical for scaling up is ultracentrifugation. An ultracentrifugation step would have to be replaced by another technique in scale-up.
Bioburden. In addition to being aware of scale-up capabilities and limitations, development personnel should design processes using techniques that minimize microbial growth. For example, if a procedure requires a buffer exchange, developers should investigate the options of performing a diafiltration or performing a dialysis at 2°8°C (as opposed to room temperature, which promotes microbial growth). Minimizing hold steps limits opportunities for microbial growth and also lessens the chance of product breakdown and aggregation. Using in-line filters during column chromatography steps helps to limit contamination from reagents.
All of our antigens are tested for microbial load before sterile filtration and filling. In almost all lots, the bioburden at that point is below the action limit, even though our processes are not meant to be sterile. Many of the buffers used during processing contain substances, like the detergent sodium dodecyl sulfate, that retard bacterial growth.
In the two or three cases where we had high bioburden in our prefiltration samples, we traced the source to a contaminated stock buffer or a container used to make the final formulation buffer. We eliminated the possibility of recurrence by making the buffer in an autoclaved container and by filtering the formulation or dilution buffer before use.
Optimal Yield. Yield does not always increase proportionally at scale-up; a tenfold increase in starting material does not always produce a tenfold increase in yield. In some cases, the yield increases at the expense of antigen purity. To maintain product quality in these cases, it becomes necessary to sacrifice yield per run. For this reason, the process development team must test the robustness of the purification procedure during the development phase.
Generally, the more of an antigen's physical characteristics (e.g., hydrophobicity, immunoaffinity, ionic strength, isoelectric point, and molecular weight) a purification process uses, the more likely it is to be successful. For example, a process in which gel filtration is the only chromatography step uses only the molecular weight of the antigen for separation. Any contaminant proteins of similar molecular weight will not be removed efficiently by such a process, and some will be left in the final product. These impurities may compromise the antigen's specificity and functionality.
Fermentation. Another key to the success of a purification process is the fermentor material. The higher the protein's expression level, the more likely the purification process is to be successful. Where the expression level for an antigen is low, the ratio of contaminants to antigen is relatively high. The process must remove a greater amount of extraneous proteins.
Process development must use the same fermentor material as manufacturing. Varying the fermentation conditions may result in the formation of different contaminants and affect the protein's attributes. Different constructs of the same antigen fermented in the same host strain can have different expression levels, different contaminant proteins, and different antigenicity. Likewise, different host strains with the same antigen construct can have different expression levels and contaminant profiles.
Fermentation also must be performed at the same scale for process development and manufacturing runs. Fermentor material from the same seed stock does not necessarily have the same expression level or behave in the same way when fermented in a shaker flask as it does in a 10-L or a 200-L fermentor.
Equipment Selection. Because the market life span of an IVD antigen may be very short and the quantities needed per year low, the use of expensive equipment and procedures (e.g., immunoaffinity chromatography) in its production is best avoided. Simpler, less expensive, and more widespread methods such as ion exchange chromatography and gel filtration chromatography usually suffice.
Automation of chromatography steps is not practical for production of most commercial diagnostic proteins because of the production scale and the low number of runs required per year. If equipment is dismantled when a process is completed (as is common in a multiuse facility), then much of the initial setup work is built into the procedure. Automation in such a case is not very practical because the amount of time saved is minimal and probably does not offset the cost of implementing and validating the automated system. Where most purification processes are performed only a few times a year and numerous processes are performed only on demand, manual techniques are more practical than automated systems.
In a multiuse facility, it is best when possible to choose equipment or components with wide-ranging capabilities, adaptable to more than one process. Doing so reduces the number of validation studies required, the number of pieces of equipment on which the operators must be trained, the number of spare parts that must be stored in inventory, and the number of vendors used for spare parts and repairs. If possible, a commercial operation should have a backup for every piece of critical equipment (e.g., centrifuges) to forestall loss of a run as a result of equipment failure. All critical equipment must be on preventive maintenance and calibration programs, as dictated by GMP requirements. Such programs should be handled by an outside department.
Validation. Another issue relevant to equipment and components is validation. Two major questions are:
* Should a piece of equipment, a system, or a component be dedicated to a particular process or validated for use with multiple products?
* Once the decision has been made to dedicate a component to a particular process, is it better to validate the removal of product carryover or to discard the component after use?
The question of whether to dedicate a piece of equipment or validate it for multiple uses depends on its cost, its frequency of use, the stage at which it is used in the process, and its interaction with product. For equipment that is very expensive and that can be used in many different processes, it is probably more cost effective to validate its cleaning than to dedicate it. This is frequently the case with equipment used in the early stages of the process, when extensive purification is to be done.
As mentioned previously, the early stages of processing in our production facility occur in small auxiliary centrifuge rooms. Dedication of equipment (e.g., bead mill, sonicator, or centrifuge) to individual processes at this stage would be expensive and pose a storage problem.
Later steps in the purification process usually involve chromatography. Because the interaction between the product and equipment and components (e.g., chromatography resin) is greater at this stage, more-innovative validation techniques are required to document cleanliness and product removal. We have found it more beneficial to dedicate the equipment and components at this stage than to validate.
Most equipment used for manufacturing (especially at later stages of purification) is too expensive to be discarded after use. Interaction between equipment and product is in most cases limited to surface contact. In such cases, validating cleanliness is not a major challenge. This is not always the case, however.
In the case of resin media, the product flows through and may interact or bind with the component. Whether to reuse or discard in this case depends on the interaction with the resin, the quantity of resin used, and the frequency of the process. For example, negligible binding occurs between a gel filtration resin that separates molecules by their size and the proteins we purify. Most processes use a lot of gel filtration media, which is quite expensive. The expense, quantity, and pass-through mechanism of separation weigh in favor of reuse of the media. Some media, such as ion exchange resins, bind protein, and validating the removal of bound proteins is a fairly extensive and expensive process. It may be more cost effective to discard these types of resins after each use, particularly if the quantities used per run are small and the annual number of runs low.
Quality control and in-process testing groups share a common goal: to ensure a consistently reproducible product. While quality control handles final product release testing and conducts stability studies for each product, the in-process group tests a variety of process intermediate samples, in accordance with GMP guidelines.
To ensure that the lot is being manufactured as specified in the batch production record, in-process tests are governed by SOPs. Analytical methods and instrumentation must be validated to demonstrate suitability for intended use.
An in-process testing group is likely to have faster turnaround times than quality control because their function is to service manufacturing. A quality control group may also be responsible for testing samples from more than one manufacturing department and therefore may have to prioritize incoming samples. Faster turnaround times are critical for minimizing hold times between steps. If in-process testing reveals problems during processing, the process can be halted immediately if necessary, limiting losses.
Specifications. The specification for an in-process assay should be tighter than quality control's specification to allow for changes that result from subsequent manufacturing steps and for lab-to-lab assay variability. For example, quality control's purity specification (by densitometric gel scanning) for a final bulk product might be set at 90%, but the in-process purity specification for pooling fractions at the final step would be higher, perhaps 95%. In-process acceptance criteria or specifications should ideally be 510% tighter than quality control's limits. In our operation, an out-of-spec in-process test result would prompt us to halt the process.
A successful process is one in which each production run consistently and reproducibly meets established product specifications. Typically, a purification process is qualified by the performance of a series of purification runs conducted according to GMPs. A minimum of three runs are needed to qualify a process at Chiron.
Analytical Methods. During the qualification period, a variety of off-line analytical methods (see Table I) are evaluated for antigen characterization. Other methods, known as in-line methods, are used for monitoring of the actual process. These include determinations of pH, conductivity, absorbance, and volume.
In-process testing generally refers to off-line analytical methods. A combination of analytical methods may be used at critical intermediate steps. For instance, fractions from a final gel filtration chromatography step are selected for pooling based on results of the following analytical methods: SDS-PAGE, HPLC, and densitometric gel scanning. The acceptable fractions to pool would be the most conservative range allowable, based on all three methods.
If a qualifying run is inconsistent with the others or yields a product of unacceptable quality, then at a minimum the one run must be repeated. When a run fails to meet in-process specifications, either the process was not performed correctly or some of its factors were not controlled. If the inconsistency of the run has no discernible cause, then the process may not be reproducible and further process development may be required. Any process that is poorly controlled or yields a product that just barely meets the established in-house specifications is likely to show problems in future purification runs.
In-process test limits must initially be broad enough to account for the inherent variation of purification processes in general, and to overcome the lack of a significant number of runs to use as a basis for the test limits. However, they must be narrow enough to control the process and to ensure the integrity of the product.
As more and more runs are completed, the in-process data should be evaluated and the specifications adjusted if necessary. Depending on the yield of the purification process and the commercial demand for the antigen, it may take several years to produce enough lots to justify tightening an in-process specification. The confidence that one can have in a process increases with the number of runs performed. For example, if the purity specification for an early chromatography step is initially 35% but the subsequent 10 purification runs pooled fractions that were all >=50% pure at this step, then the specification should be tightened to reflect the actual results.
In-process testing data are continually trended and evaluated in order to assess the quality of the product and the reproducibility of the manufacturing process. Examples of trend analysis include percent recovery and total protein values for specific intermediate steps and for the final bulk. These data are especially important for determining run-to-run variation. Some in-process testing (such as SDS-PAGE on extract, pellet, and wash samples) is done to collect historical information that is helpful for troubleshooting in the event of product- or process-related problems.
Stability Studies. Performing short-term stability studies for new antigens is prudent to ensure that the formulation of the product, as well as storage conditions, do not result in product breakdown or aggregation/precipitation. Rarely is this information available before transfer. Because development material is often in short supply, material from the consistency series can be used for these studies. A short-term stability study might address the following questions:
* What is the stability when the product is stored at 20°C compared with 60°C at various time points (for hold steps)?
* How well does the product hold up when frozen and thawed several times?
* How does it do when heated to 37°C or to boiling?
* How long is it stable when stored at 2°8°C, or at room temperature on the bench?
* How well does it handle being thawed quickly? slowly?
* Does it break down when spiked with a heavy load of microbes typically isolated in the manufacturing environment?
As soon as the antigen-purification-consistency series has been deemed successful and all initial processing parameters and final bulk antigen specifications have been established, the antigen needs to be entered in a long-term stability study, under the oversight of the quality control department. This study should ideally be initiated immediately following the consistency series in order to gather data for the longest time before introduction of the product into the market.
Process and product segregation, process transfer and scale-up, and in-process testing are important considerations for any multiuse protein purification facility. Manufacturing personnel involved in protein purification regularly encounter the problems discussed here and more. The setup, procedures, and processes we have shared provide insurance against many of these pitfalls, and tools for overcoming others.
Dan Gilbrech is associate manager of diagnostics purification, Fay Kim is associate manager of diagnostics analytical purification, and Cheryl Covert, MBA, is associate director of diagnostics purification at Chiron Corp. (Emeryville, CA).