Characteristics of a Good Antibody Manufacturing Process

Bradley Wolk, Paul Bezy, Ray Arnold, and Greg Blank

Reprinted with permission from BioProcess International 1(9):50-58 (September 2003)

ver the past five to ten years, manufacturing of antibodies for use as therapeutic agents has evolved from a focus on the art and science to a focus on robust, compliant, manufacturingscale operations. Laboratory-scale purification processes do not necessarily translate directly to robust and reliable processes in the manufacturing environment (1). For that reason, it is critical for a company to determine what constitutes a good manufacturing process and factor those attributes into early process development activities. Each company, and even different manufacturing facilities within the same company, may place different values on specific process attributes. Integration of those ideas into process development is vital to successful large-scale or commercial production. Addressing potential operational issues during process development is much simpler and more effective than trying to change an approved, validated commercial process. The ideas discussed herein apply to manufacturing in general, but the primary focus of the examples and descriptions is on the recovery process. Defining Characteristics of a Good Manufacturing Process: A good place to start is with current commercial or clinical processes. Evaluations of such processes by the manufacturing and process development groups will help identify technical, operational, and compliance related issues. Typically, those issues can be grouped into a few categories that are key contributors to success in
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manufacturing. They will need to be prioritized and potentially balanced against each other so that guidance can be given to the process development group regarding processing preferences. Many processing options exist, and examples of several typical processing trade-offs are discussed below. Communication of these ideas to the entire process development organization is another critical component of this effort.

PROCESS GOALS

Two types of process goals are important: product-specific goals and generic process goals. Product-specific goals have been the traditional focus of process development efforts. They include achieving maximum yields, adequate purity, desired logs of viral clearance, target bulk product concentration, adequate product stability, cost of production targets, campaign production targets, and adherence to manufacturing schedule. Those goals are critically important, but they are quite different from the generic process goals that help define a good antibody manufacturing process. Seven categories of generic process goals have been established. Many authors have described similar categories as they apply to process development and transfer into manufacturing (25). The categories are not exclusive and are often closely related. In fact, some goals may conflict with others in specific circumstances. Process development responsibilities must include

consideration of all these attributes in early development: safety, compliance, plant capacity, ease of operation, cost of production, process characterization/validation, and robustness. Safety: Each process should be designed to help maintain a safe working environment. A safe process minimizes personnel exposure to hazardous chemicals, process waste or byproducts, and dangerous operational practices. Concentrated acids and bases can be minimized in a process by preparing buffers without a pH titration (Table 1). Use of conjugate acids and bases can aid in accomplishing this goal. Buffer choices can affect manufacturing in other ways as well. Raw materials that are difficult to break up or get out of drums (hygroscopic materials, for example) could contribute to unsafe practices. Understanding the disposal requirements (local, state, and federal) for materials that are considered to be hazardous wastes and limiting the use of these materials also contributes to the overall safety of a process. Hazardous waste collection is resource intensive, and disposal can be quite expensive. New equipment required for manufacturing must be designed with safety as a key consideration. That includes ergonomics, insulation to protect technicians from hot process lines, control of noise levels, sampling methods and devices that minimize exposure, lifting-assist devices for handling heavy equipment, adequate

wheels on equipment for the surfaces involved, and appropriate seismic considerations. Moving, operating, and servicing should be evaluated as part of the overall design of new equipment. Compliance: Regulatory guidelines for biotechnology production place an emphasis on processes running as they were designed, with product release implications for deviations from normal practice (6). Because cleaning is integral to all operations, equipment design needs to incorporate best practices for effective cleaning, chemical sanitization, and steam sterilization. Reliable equipment is critical for a process to meet operational requirements routinely. Corrosion and bioburden issues can negatively affect production and product release, so processes and equipment should be designed to limit those risks. Proper specifications for materials of construction, fabrication practices, operational practices, buffer components, and buffer pH all contribute to prevention of problems. Materials of construction considerations include chemical compatibilities, temperature tolerances, mechanical properties, extractables, and contact time with the process. Process parameters must be appropriately designed, with ranges narrow enough to be effective and wide enough to be achievable in manufacturing. Plant Capacity: A good manufacturing process will help maximize productivity. Plant throughput is a function of how much time, how much equipment, and how many people are required to operate the process. Designing a process to require fewer steps, fewer tanks, or fewer buffers will improve plant productivity. Higher yielding processes and higher success rates both help to reduce the number of runs that will be required to meet production goals, thus having a positive impact on plant capacity. Process steps of long duration relative to other steps may reduce capacity by creating process bottlenecks.
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Specification of highly reliable, standardized equipment that can be used for multiple products will help reduce downtime, training requirements, spare parts inventory, documentation requirements, and equipment validation. Automation of a process can have major impact on plant capacity. When used appropriately, automation can improve capacity by increasing success rates, reducing labor requirements, and shortening equipment turnaround times. However, when automation systems are too complex or require unscheduled downtime, they can have a negative impact on plant capacity. Specification of room-temperature operations will help eliminate bottlenecks caused by limited coldroom space and reduce equipment downtime (especially electronic components) caused by cold-room conditions. Ease of Operation: Simple, reliable process equipment and solution preparation are major factors in the ease of operation for manufacturing. Simple buffer preparation, without the need for titration, facilitates operating any process. Weight- or volume-based additions are much easier to perform than titrations. Standardization of processes using similar equipment, procedures, documentation, raw materials (including filters and resins), and automation structure will improve manufacturing success rates. Process development efforts should include evaluation of equipment sizes, buffer volumes, solids handling, solvent requirements, column packing, and hazardous waste issues to ensure that the process and the equipment needed to operate it are appropriate for the manufacturing facility. Simple items such as elevator dimensions, door widths, and equipment weight can lead to major operational issues if not addressed before the equipments arrival on site. Cost of Production: A good process will be cost effective, and alternatives to reduce costs should be evaluated. This is not to say that the cheapest process is the best. An understanding of raw material costs,

resin and membrane lifetimes, process yields, labor requirements, required process steps, utility costs, hazardous waste disposal costs, and other variables can be analyzed together to help make informed decisions about the cost of production. A high success rate will lower the cost of production, reducing the number of runs required. And a more expensive chromatography resin that improves yield may help lower the cost of production. Careful analysis of the cost of production will show the relative contributions of cell culture, purification, and fill-and-finish operations to the overall process cost. Process models or simulation tools can be extremely valuable for this type of analysis (7). When such analysis is done early in development, it can be a valuable tool for determining focus areas for process improvements. In some cases, the fill-and-finish component may be a major contributing factor to cost, indicating that extra resources allocated to improve purification yields may not be warranted. Process Characterization and Validation: A key feature of good manufacturing processes is adequate process characterization and validation. These are required to support robust, compliant process operations. Process validation is a requirement of the FDAs Current Good Manufacturing Practices Regulations for Finished Pharmaceuticals (21 CFR Parts 210 and 211). Guidelines on both validation and characterization are available from the FDA (810). These studies will help define operating targets and acceptable ranges for the process. Typical characterization/validation studies include membrane and resin lifetimes, in-process hold times, buffer hold times, protein load limits for columns, pH and conductivity specifications for buffers, extractables and leachables from resins and membranes, virus removal/ inactivation, impurity removal, and small molecule clearance. The combined results of the studies will

establish target and acceptable ranges for process parameters in batch records and regulatory filings. It is advisable to identify critical process parameters during process characterization activities and validate the acceptable ranges of those parameters before filing a biologics license application (BLA). Definition of reasonable ranges for process parameters, reflecting the ability to control them (e.g., pH 5.0 0.2 rather than pH 5.0 0.05), will have a major impact on the success rate of a process in manufacturing. A buffer specification that is too narrow will lead to unnecessarily rejected batches of buffer, increasing labor requirements and negatively affecting plant capacity. Robustness: For the purpose of this discussion, process robustness is defined as insensitivity to small changes in process parameters or conditions, and the process is simple and reliable to operate. Robustness is the most important consideration when deciding whether a process qualifies as a good manufacturing process because it affects most of the other six attributes. A good, robust process will be characterized by the following general descriptions. Relatively Insensitive to Temperature: A process should be able to handle temperature deviations consistent with the temperature control capability in the facility. Reasonable Hold Times Consistent with Manufacturing Operations: A 30minute hold time (15 minutes) may be too short to handle normal operational delays. Designed to Handle Normal Process Variability: Fermentation yields, product variant levels, mass loading onto columns, and chromatography pool concentrations will vary from run to run in manufacturing. Relatively Simple Automation Transition Conditions: Decisions for the automation system should be based on simple, reliable instrument signals or calculations. Complex equations used to control automated systems are difficult to test and validate. Elution decisions based on volume and/or optical density measured by absorbance at 280 nm
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(A 280) are more robust than using the first derivative of the A 280 signal. Instrument Ranges and Accuracies Considered in Developing Specifications: In-line sensors for pH, conductivity, and A 280 are typically userstandardized probes checked with other user-standardized devices. Manufacturers of such sensor/ transmitter pairs will specify instrument accuracy as a percentage of full-scale readings. Based on those accuracies, small errors from process targets can occur during normal operations. The cumulative effect of those errors must be considered in the overall design of a process. For example, dry powders for a buffer will be weighed and batched into a make-up tank (deviation #1). Following dissolution of the powder, water addition to the final batch volume will be measured by a level probe, batching meter, or load cell (deviation #2). Testing of the buffer after preparation may involve a pH or conductivity measurement along with the associated error (deviation #3). Table 2 lists some common-sense values to use for manufacturing specifications. Consistent Buffer and Raw Material Specifications: Problems can occur if the endotoxin specification for a buffer solution is lower than the endotoxin specification for either water or the dry buffer components. Specifications Allow for Normal Variation Between Analyses: Variation can be introduced from three sources: the process, sampling, and analytical testing (11). Redundant tests on samples may therefore give different results. A good approach is to specify a wider range for subsequent analyses. For example, a chromatography equilibration operation with a pH 6.0 0.1 buffer specification would have a wider precolumn specification on the same buffer sampled just before the column (pH 6.0 0.2) and an even wider column effluent specification (pH 6.0 0.3). Figure 1 illustrates this concept. Appropriate Tests for Filter and Column Integrity: The integrity test method for filters and chromatography columns should be extremely reliable.

False integrity test values can create a great amount of unnecessary work in manufacturing. Filter integrity tests should be able to determine whether a test is invalid. Specifying the air diffusion rate at less than 50 mL/ min would indicate a value of 0 mL/ min as a passing result. Because this no-flow condition could be caused by a closed valve, a more useful specification would be greater than 5 mL/min and less than 50 mL/min. Chromatography column bed integrity tests should identify packing issues that adversely affect the separation (e.g., cracks and headspace problems). Online transition analyses have proven useful in identifying these types of issues (12). Process Parameters Evaluated for Effects on Overall Performance: Changes in the operation of a chromatography step could affect the overall ability of a manufacturing process to achieve the required product purity. For example, elution conditions on a protein A column can be adjusted to increase the yield of that specific unit operation, but a higher level of host cell proteins might be captured in the pool. Subsequent chromatography steps would need to be evaluated to ensure they have the capacity to remove additional host cell proteins before such a change is implemented. Include Options to Back-Titrate Pools: Titration will allow continued processing in the event of minor pH or conductivity deviations. Evaluate Differences Between Manufacturing Floor and Laboratory Instruments: High A 280 values measured on instruments with different path lengths could create a few surprises in manufacturing. Instruments from different vendors can give different values (e.g., A 280, turbidity) when measuring the same solutions. pH Adjustment Solutions Have Sufficient Buffering Capacity at Target pH: A poor choice of buffer for use in pH adjustment could require large volumes, diluting the pool and affecting downstream operations. A poor choice of buffer ingredients can make certain pH targets difficult to achieve. Figure 2 illustrates this.

Identify Critical Mixing Operations: Mixing is often taken for granted during creation of a manufacturing process. Mixing solids that are difficult to dissolve or mixing liquids with very different densities involves specific design considerations. Poor mixing can result in localized conditions of unacceptable ionic strength, pH, or viral inactivation. Mixer speed, when high enough to cause air entertainment, might affect protein stability. Raw Materials, Filters, and Resins Come from Reliable Vendors: Such materials must have appropriate specifications. Even robust processes are difficult to operate if the raw materials used are not of consistently high quality. Buffer salts incorporating even small amounts of debris can cause major problems in operations.

PROCESS TRADE-OFFS

Process development often involves balancing the seven characteristics discussed above with each other. A higher cost of production may be necessary to improve robustness, for example. Several examples of process design trade-offs in a recovery process are presented here to illustrate the importance of understanding key factors to consider in process development for each facility and organization. Example #1 Chromatography Column Equilibration: A single equilibration/wash buffer can be used to equilibrate a column, but a large volume is necessary. Instead, a twobuffer system might be used (Table 3), in which a more concentrated solution is used to do a partial equilibration followed by a second buffer that completes the job. That two-buffer system uses less total volume (and time) but requires preparation of the second buffer. Considerations include buffer tank availability and size, buffer costs, process time, and labor requirements. Example #2 Urea or Filtration for Viral Reduction: To obtain the required logs of viral clearance less than one theoretical viral particle per one million doses (13) in a purification process, urea or viral filtration may be implemented. Urea addition requires the urea solution to

be made, deionized, and stored cold. Because of reionization, the urea buffer has a relatively short expiration time. Urea is a difficult solid to handle and requires assembly of a deionization system. It also may require special waste disposal. Holdtime validation studies must be performed to evaluate viral inactivation versus time and check for protein damage. A short maximum hold time could be difficult to achieve routinely in manufacturing. Viral filtration is very similar to other standard filtration operations. It constitutes another process step, requiring buffer preparation, equipment cleaning, integrity testing, documentation, and training. Single-use filters are relatively expensive and are characterized by low flux. Filtration of large pools is therefore time-consuming and expensive. Considerations include equipment and labor availability, plant capacity, process time, waste handling, and cost of production. Example #3 Chromatography Pool Conditioning: To obtain the correct pH and conductivity for a subsequent chromatography step, the pool is conditioned with a buffer added to the pool tank. This conditioning adds another step to the overall process, requires an additional tank, adds sampling and documentation requirements, and increases bioburden risk. Because the addition will change pH and conductivity of the pool, elution conditions on the preceding chromatography step can be less constrained. Another option is to elute the preceding column at conditions that allow direct load onto the subsequent column. That would eliminate the additional step but may narrow the acceptable conditions for elution or affect subsequent purification steps. Considerations include process robustness and time, labor requirements, tank availability, and compliance. Example #4 In-Process Decisions Compared with Laboratory Testing: A chromatography operation is automated, and the elution is controlled by online optical density

(OD) instrumentation and volume totalization. That allows simple, effective collection of a product pool. Another possibility is to fractionate the pool and use laboratory analysis to determine which fractions to combine into a final pool. That method would allow decisions to be made using product-specific assays but is time and labor consuming, and it poses more of a bioburden risk. Considerations here include tank availability, turnaround time for the laboratory assays, process robustness, ease of operation, compliance, and process characterization/validation. Example #5 In-Process Hold Times: Hold times for product pools can be critical if conditions such as low/high pH or the presence of urea cause product change. Very short maximum hold times will allow better control over product quality but may constrain other operations and be difficult to achieve if equipment-related issues arise. Delays in analytical tests also could make short maximum hold times difficult to achieve. Longer hold times are easier to meet routinely but may affect product quality and plant capacity. Considerations here include ease of operation, process robustness, plant capacity, and process characterization/validation. Example #6 Process Validation: Urea is used for viral inactivation and has been validated to deliver 3 logs of viral inactivation in 3 M urea for 30 minutes. Process validation was done using those exact conditions, with no ranges around the setpoints. Because of errors associated with the urea preparation, the final concentration may be below 3 M, which is not covered by process validation. Additional validation studies could be done at a number of urea concentrations and hold times (Figure 3). That would add cost and time to the testing program but would allow for a more robust operation in manufacturing. Considerations here are process robustness, characterization/ validation, and compliance. Example #7 Long-Term Storage of Membranes and Resins: Membranes and resins can be stored between production campaigns and
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reused later (with the proper validation studies). This practice can save money because new membranes or resin are not required as often. Based on resin volumes or filter areas, the raw material cost, and the length of campaign, cost savings could be high. But a substantial amount of work might be required to implement the practice: validation of long-term storage conditions, design of special containers for long-term storage, a tracking program for membranes and/ or resins in storage, and allocation of appropriate storage space. Considerations in this case would be cost of production, compliance, and process validation. Example #8 Multiple Concentration Steps to Reduce Volumes: Tangential-flow filtration (TFF) can be used at various points throughout a process to concentrate the pools, which potentially reduces buffer volumes, tank volumes, and/or column volumes for downstream operations. Volume reduction could make it possible to operate a process in a facility that is too constrained to handle the full volume process. The additional TFF steps would require cleaning, steaming, documentation, equipment, and process validation and they might increase yield losses. Considerations in this case would be cost of production, plant capacity, and ease of operation.

be incorporated into the earliest phases of process development possible. Collaboration between manufacturing and process development groups enables new processes to be consistent with an organizations manufacturing capabilities and standards, which helps companies raise the success rates of their manufacturing processes. An assessment mechanism should be developed to quantify the extent to which each new process meets the agreed-upon goals.

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We will, that is, if you send news of product releases (or setbacks), clinical trial results, research breakthroughs, advances in technology, association and worldwide government initiatives, economic development initiatives, and meeting reports to assistant editor Molly Pumper at mpumper@bioprocessintl.com.

ACKNOWLEDGMENTS

The authors would like to thank Adeyma Arroyo and Jerry Cacia for their contributions to this article.

REFERENCES

COLLABORATION IS CRITICAL

Each organization should specify its own process goals and criteria used to determine what constitutes a good manufacturing process. They may differ between different manufacturing sites. Ideas like the above should be discussed by the manufacturing and process development groups so that they can

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Reviews in Biotechnology 1993, 13, 3, 195253. 6 Center for Drug Evaluation and Research. Compliance Program Guidance Manual for FDA Staff: Drug Manufacturing Inspections Program, 7356.002, US Food and Drug Administration: Rockville, MD, 2002. 7 Petrides, PP; Koulouris, A; Lagonikos, P. The Role of Process Simulation in Pharmaceutical process development and Product Commercialization, Pharmaceutical Engineering, January/February 2002 , 5665. 8 Center for Drug Evaluation and Research. Guideline on General Principles of Process Validation. US Food and Drug Administration: Rockville, MD, reprinted February 1993 (External Web site ). 9 Center for Drug Evaluation and Research; Center for Biologics Evaluation and Research. Guidance for Industry, Q7A Good Manufacturing Practice Guidelines for Active Pharmaceutical Ingredients. Fed. Regist. 2001, 66 (25 September), 49,02849,029. 10 Center for Drug Evaluation and Research; Center for Biologics Evaluation and Research. Guidance for Industry, Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. Fed. Regist. 1999, 64 (18 August), 44,928 44,935. 11 Box, G; Hunter,W; and Hunter, JS. Statistics for Experimenters. John Wiley and Sons: Hoboken, NJ, 1978, pp. 571583. 12 Larson, T; et al. Use of Process Data to Assess Chromatographic Performance in Production Scale Protein Purification Columns. Biotechnology Progress, 2003, 19, 485492. 13 Center for Drug Evaluation and Research; Center for Biologics Evaluation and Research. Guidance for Industry, Q5A Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin. Fed. Regist. 1998, 63 (September).

Bradley Wolk is a principal engineer and senior group leader in recovery sciences, Paul Bezy is the senior manager of the recovery sciences pilot plant, Greg Blank is the director of recovery sciences, and Ray Arnold is a senior manufacturing technical specialist in global manufacturing sciences and technology at Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, 650-225-3134, fax 650-225-4049, wolk.brad@gene.com.

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