Advances in upstream processing of monoclonal antibodies (mAbs) have resulted in increasing titers, cell densities, and other properties that have challenged existing downstream processing operations. It is essential when developing downstream mAb purification processes to understand the product and process goals and focus on platform solutions that offer the greatest throughput.

Advances in both upstream and downstream technologies are helping to facilitate downstream processing of mAbs, but companies need to use a more holistic approach to process development and pursue various avenues of process intensification if real progress is to be made.

Before any process development work is completed, it is imperative, asserts Louise Duffy, senior VP and head of scientific project leaders at Abzena, to first understand exactly how the mAb is constructed and how it will work clinically.

Developers should start by identifying their product quality goals, driven by critical quality attributes, along with process requirements, facility capability, product requirements, and cost of goods, agrees Natraj Ram, vice-president of innovation, bioproduction at Thermo Fisher Scientific.

Considering the product stage (early or late) is also critical to ensure which aspects are prioritized, so that the appropriate level of process understanding and control strategies are achieved during development, according to Ram. Identifying the target scale for the commercial process will also help determine the scale and technology choice and transitions throughout development, he adds.

The next step, Duffy says, is to develop the downstream process to enhance the purity of the desired mAb while reducing product and process related impurities. That should be done, says Kevin Brower, global head of purification development–mammalian at Sanofi, with a defined platform set of raw materials and a platform approach for performing development.

“Using this approach is the most critical best practice because it provides structure for all process development scientists in their work at the bench as well as structure for interactions across the manufacturing network for tech transfer,” Brower states.

Understanding the tools needed to identify product-related impurities while developing purification is also key, according to Duffy. “These two activities must be done collectively with assay and downstream capability developed collaboratively,” she insists.

Use appropriate resins, filters, chemicals, and single-use components

The ability to achieve high throughput is always an important objective in a production area. For downstream process development, that includes higher flow rate, more rapid turnaround, and more streamlined processes, according to Haiou Yang, director of downstream process development for Avid Bioservices.

For downstream process development, the appropriate choice of resins, filters, chemicals, and single-use components is critical to the success of scale up and manufacturability, adds Ram. Potential requirements for chemicals should be considered depending on the target markets for the mAb product, while resins selection should take into account scalability if a chromatography process will need to be scaled from 1-L to 200-L columns. Assurance of integrity and access to standardized connections are two important factors that should drive the selection of single-use (SU) components.

The strategy for downstream processing for a particular mAb is dependent on the specifics of the upstream process, the capabilities of the facility, and the available equipment for the various unit operations required for mAb purification and final formulation. In general, each unit operation is sized based on the amount of mAb to be processed, factoring in the recovery of each step as it goes down the sequence.

Overall, the downstream processing capacity is the amount of upstream material that can be processed in a certain amount of time. Sanofi currently uses a mix of in-house Excel calculations and external software to model downstream capacity, identify bottlenecks, and guide its tech transfer activities, according to Brower. He notes that these methods are pretty well-established in the industry.

There are two possible strategies for determining the overall downstream processing capacity, says Ram. The first involves determination of the process throughput for each unit operation, choosing technologies that provide the best throughput considering the operational constraints at scale.

The second option is particularly relevant for batch operations such as mixing and filtration, according to Ram. “In these cases, both throughput and the ease of transitioning from one step to another are both considered and the choice of [single-use] SU technologies thus makes a difference,” he observes.

Having appropriate mixing systems with the right bags, filters, and sensors can help with combining multiple steps in one, Ram explains. For example, he notes that performing the inactivation and neutralization steps with a single mixing system that has a pH sensor built in and is equipped with appropriate tubing to pump in and pump out the material will help with efficient processing.

High titers from upstream can either be processed through scaling up processing equipment, which may be limited for some facilities, or through multiple cycles, which can extend processing time, according to Gene Yoshioka, senior director of manufacturing at Avid Bioservices.

Assessing process capacity up front saves time and resources and establishes feasibility, Duffy says. “Performing upfront work to characterize maximum binding capacities, retention times, and equipment scales becomes critical to optimizing the downstream process to match with the upstream titer,” Yoshioka agrees. Designing a process with the capacity of the existing facility in mind also facilitates a smooth technology transfer and enables early production planning.

The typical approach to estimating capacity for a downstream process, explains Duffy, is to look at the titers in the bioreactor upstream, and then size up first steps to hit the sweet spot between not spending too much on resin while allowing for a reasonable number of sub-batches and managing time for the operations. The best strategy, she asserts, is to map the entire process, perform a full mass balance across the whole process, and determine where hold steps may be needed/wanted and then work out how to develop data to support those hold steps.

Internal collaboration is beneficial for this approach, Duffy adds. Process development teams can discuss the feasibility of the process and operational teams can look at more pragmatic issues such as fit with current facilities.

Titer is not the only aspect to consider, though. “During upstream production,” observes Ram, “if too much focus is placed on product titers, other factors such as cell concentration, cell viability, and various product quality characteristics may be impacted. These upstream factors will most likely impact the subsequent recovery and purification process steps.”

Indeed, in recent years there has been a greater focus placed on developing strategies to match downstream technologies with high-titer upstream processes, according to Yoshioka. He stresses once again that it is not just an issue of process capacity. With media and cell lines that are able to maintain higher cell densities, there is also an increase in process impurities (i.e., DNA, host-cell proteins, charge variants, etc.) that the downstream process needs to be designed to remove.

Examples of more traditional improvements helping to eliminate bottlenecks noted by Brower include higher capacity resins and membranes, as well as single-pass concentrators. Ram adds that adoption of higher capacity, lower cost resins, and membrane technologies is allowing manufacturers to increase facility throughput without retrofitting. However, he also notes that it is important to develop resins that provide better capacities, resolution, and throughput.

Advanced depth filtration technologies, meanwhile, enable the harvest of large volumes quickly and easily while providing the benefits of a more clarified feed stream going into the purification process, according to Ram. “Because these technologies combine chemistries with carefully designed matrices that provide size-based separations, these technologies go beyond improving throughput and provide higher quality product,” he says. They also allow for easier scale-down modeling compared to centrifuges, reduced cost of goods at scale up, and lower volume pools. The latest multi-mode versions under development make it possible to have smaller volumes, less dilution, and higher yields, Ram says.

Beyond advances in technology for specific unit operations, Ram points to the use of multi-column chromatography, integrated flow-through steps, continuous inactivation and neutralization, and in-line dilution of buffers as important downstream technologies that are enabling downstream processing to catch up to the higher levels of upstream productivity.

Brower also believes that high-throughput process development approaches allow mAb developers to better understand and characterize their processes than was possible five to 10 years ago. “Access to this greater knowledge leads to more robust process outcomes that can better handle the variability provided downstream by various forms of intensified upstream processes,” he explains.

Single-use technologies are having a major impact on how downstream processes are developed as well, according to Duffy. She notes that developers can more easily explore changing the order of process steps, or introducing new ones, depending on how a molecule is behaving and what the impurity profiles look like. “This greater flexibility allows for more rapid fine-tuning of processes,” Duffy says.

Yang cautions, though, that while the industry has been working to adapt to new devices and technologies, the process of converting to completely new/different techniques and instrumentation requires time and careful execution. “It is important to note that the easier a technology can be implemented, the faster the industry is able to adapt to it,” she comments.

Despite numerous advances in downstream processing technologies, there is still a disconnect between upstream and downstream processing, according to Duffy. “Firstly, in the last five to seven years, upstream titers have almost tripled, while downstream capacity has not necessarily matched pace. Secondly, some downstream technologies are still very manual and laborious to run,” she explains.

To address this issue, a holistic view of the entire manufacturing process is needed. Process developers, Duffy asserts, must proactively think about the impact of upstream decisions on downstream processing to fully understand what technologies will be required for downstream processing.

A holistic approach that considers both upstream and downstream processes is critical to success,” agrees Ram. “While these two manufacturing processes rely on each other to be successful, they are frequently developed separately. Developing them in parallel, however, can help meet aggressive program timelines and allow for inherent synergies. Harmonizing the processes enables greater understanding of the upstream challenges for the protein of interest being produced, which can then help negate possible problems encountered later in the development process,” he explains.

Process intensification is necessary, too

Given that most downstream steps are inherently mass based (process a certain amount of product), Ram asserts that efficiencies can only be gained through integration or intensification of these steps. “Many of the technology advances made to date have the potential to improve productivity or throughput, but until they are integrated to allow for synergistic benefits, much of the advantage cannot be realized,” he says.

While initial efforts have occurred, integration of unit operations from both operational and control standpoints has yet to be fully achieved and will require collaboration across the industry to bring harmonization of components, equipment controls, and software platforms, Ram comments.

In the near-term, agrees Brower, application of integrated and continuous (or connected) process operations will be the key to improving the productivity, long-term sustainability, and flexibility of downstream processing in the biopharma industry. “Such approaches,” he concludes, “have an impact on space requirements, increase the ability to apply single-use technologies, drive down cost of goods to increase patient access, and allow manufacturers to dynamically respond to changes in treatment approaches and reimbursement strategies.”

The need for process intensification and continuous processing will, in the long term, drive the development of process analytical technologies (PAT) beyond basic sensors that will allow for the enhancement of throughput with better process control for both continuous and batch processing, according to Yang.

“Novel purification formats (non-chromatography, flow-through technology) and the incorporation of PAT tools to enable real-time release and forward processing to drug product,” agrees Brower, “will lead to a further step change (leap) beyond the current standard approaches.”

Meanwhile, Ram notes that advances in single-use centrifugation or related technologies that reduce the burden on harvest filters could be a significant improvement to address challenges posed by increasing cell densities and higher titers in upstream processes. Integration of multicolumn chromatography skids with buffer blending capability will significantly improve the efficiency of this operation. There is also, Ram says, still great opportunity for the development of resins that can provide high resolution while also offering high capacity and throughput. He also notes that future single-use systems that can accommodate a wide range of scales will also help reduce overall capital costs for multiproduct facilities.

Another major opportunity for improvement in downstream processing, according to Brower, is less related to upstream advances and more related to the diversity of molecules in the biopharma pipeline. “This diversity of modalities presents unique purification challenges for which we are developing solutions and approaches across the industry,” he says.

Cynthia A. Challener, PhD, is a contributing editor to 

When referring to this article, please cite it as C. Challener, “Defining a Holistic Approach to mAb Purification Processes,” 

Articles

Cover Story

Understanding the process and product goals is just the first step to a holistic approach to process development.

Defining a Holistic Approach to mAb Purification Processes

Testing for viral clearance in downstream bioprocessing is growing increasingly challenging for new biologic modalities as the processes to make these therapies are themselves becoming more complex. For example, gene therapies rely on the use of viral vectors in the manufacturing process, so distinguishing viral vector components from virus contamination requires sensitive and discriminating testing methods. Do current techniques address these challenges or is further innovation required to ensure continued product quality and purity?

There are two primary factors that can increase the difficulty of viral clearance, says Alex Schwartz, product specialist, Viral Clearance, Sartorius. First, the raw material inputs for a number of these new biologic modalities require the use of high-risk items, such as animal- or human-derived sera, he explains. The use of these high-risk materials means there is a significant increase in viral contamination risk because these materials are frequently contaminated, and testing is not always 100% effective at screening, he cautions.

Secondly, these new modalities have significantly reduced downstream unit operations. “While viral vectors cannot be processed through traditional viral inactivation methods, they often only rely on one or two column processes that may or may not provide robust viral clearance greater than four log reduction values (LRV),” Schwartz states. Thus, in cell therapies, there are no effective chromatographic techniques, and, like the viral vector space, no viral inactivation is considered safe for the product. Additional efforts are thus usually taken as a viral barrier in the cell culture processing, he adds.

Marc Breton, group leader III, Viral Clearance Services, Eurofins BioPharma Product Testing, adds that, for these new modalities, the challenge to overcome is to accurately distinguish native (product related) virus from the spiking virus used to challenge a process step.

Meanwhile, Joshua Huffer, technical specialist, Field Technology Management, MilliporeSigma, emphasizes that the most difficult aspect of many of these advanced medicinal therapeutic products (ATMPs) is the fact that it is difficult to find a processing step that will selectively inactivate or remove the potential viral contaminants without inactivating the biological product. “For example, retroviral or lentiviral gene therapy vectors will be inactivated by steps that inactivate enveloped viruses. These products are relatively large, so that virus filtration cannot be used,” he points out.

The same difficulties also exist for cell therapies. Apart from non-enveloped viral therapies (e.g., adeno-associated virus [AAV] viral vectors), viral clearance is not an option for cell and gene therapies, Huffer says. “In the absence of any potential viral clearance, extra scrutiny is placed on the cell line characterization and testing the input raw materials to reduce the risk of any potential viral contamination,” he explains. “For enveloped viral vectors and cell therapies, we recommend the use of barrier methods to treat the cell culture medium and medium components.”

Methods, such as high-temperature, short-time (HTST) and virus reduction filtration, reduce the risk of the potential entry of a viral contaminant into manufacturing, Huffer adds, explaining that barrier filters are designed for this purpose. “This mitigation method is well-suited to the smaller batch sizes used in cell therapies and some gene therapy products,” Huffer states.

Meanwhile, non-enveloped viruses, such as AAV, can follow more traditional viral clearance routes. “These viruses are much more resistant to detergent and low pH treatment steps, which will inactivate enveloped viruses. The use of viral filters with larger pore sizes (e.g., 35 nm, 50 nm, 70 nm) will allow the small AAV vector to pass through, while retaining larger enveloped viruses. An inactivation step and a viral filtration step will add robust clearance of enveloped viruses to the manufacturing process for an AAV product. Affinity chromatography and anion exchange chromatography can potentially contribute to viral reduction from the purification stream,” Huffer explains.

With these challenges, having the right approach and techniques is important for successful virus removal to ensure product quality/purity for ATMPs. Although the number of chromatography steps for a gene or cell therapy are limited compared to other fields, such as monoclonal antibody (mAb) products, there are still common steps that can be used for most gene therapy studies, including affinity and anion exchange chromatography, says Tareq Z. Jaber, PhD, manager, Process Evaluation, Charles River Laboratories. Jaber explains that these steps can cover both enveloped and non-enveloped viruses, including parvovirus models (in AAV products) or retrovirus models (in lentiviral products). “Including other steps, such as detergent inactivation, will depend on the product nature itself. If AAV is used, detergent can be applied since AAV shows resistance to detergent inactivation while retrovirus or herpes virus vector products will be sensitive to detergent,” he states.

Polymerase chain reaction (PCR) techniques are useful for titering virus concentrations in these complex systems—especially for gene therapy products, which have potential native virus that could interfere or even obscure the result in an infectivity assay, adds Breton. “Due to their specificity, PCR techniques can solve this challenge, but are unable to distinguish between active and inactive virus. To overcome this challenge, analyzing virus titers determined by both PCR and infectivity techniques can be helpful, depending on the process step being challenged,” he says.

For an AAV vector, an enveloped inactivation step and virus filtration using a large pore filter will usually provide effective enveloped viral clearance, Huffer says. “For cell and enveloped vector gene therapy products, viral clearance steps are not feasible and, therefore, selecting and testing the cell lines and raw materials used in the manufacturing process are key to reducing the risk of potential viral contamination. The use of a barrier method to prevent entry of a viral contaminant into the process stream should also be a key consideration,” he states.

Timeline constraints are another challenge that tends to be a common concern for viral clearance testing, regardless of product type, Breton says. “I always advise that early communication with your viral clearance vendor is key to the quality and performance efficiency of any viral clearance study—this is doubly important with novel techniques and products,” he says.

“Time is always a concern, especially these days,” Jaber agrees. He points out, however, that for clinical Phase I/II-stage programs, regulatory agencies do not require viral clearance for all gene therapy products studies, although the agencies may recommend it. “If there is no risk of copurification of a helper virus or of an adventitious virus (e.g., from the cell line, such as an endogenous retrovirus or rhabdovirus, or from raw material) agencies do not require viral clearance studies. However, it is regarded mandatory to analyze for Phase III studies and commercial release,” he states.

If the downstream process has clearance potential (chromatography steps, for example), the clearance should be analyzed to demonstrate safety of the product, Jaber adds. “When required (in case of a known contaminant or copurifying virus), viral clearance should be adequately demonstrated as per the recommendations in ICH [International Council for Harmonisation] Q5A (1),” he says.

Huffer adds that for time-sensitive therapies, such as autologous cell therapies, viral clearance steps are not an option. “Rapid detection of potential contaminants—viral and microbial—is critical for these time-sensitive therapies. Making use of rapid testing options, such as rapid sterility, quantitative polymerase chain reaction (qPCR), or other molecular methods for detection of mycoplasma and other known contaminants, are valuable techniques to ensure patient safety,” he states.

Schwartz adds that, since viral clearance should still be viewed as a platform approach to cell therapies, most viral clearance claims should not impact patient material or their specific timing needs. “Outside of viral clearance, what can make an impact is the speed of QC [quality control] virology testing required to return patient cells to the clinic,” he says.

The need to improve viral clearance for these new biologic modalities requires innovation in thinking as well as in technology. Inactivation and filtration steps used for enveloped viral clearance in AAV manufacturing processes are not new but can still provide robust viral clearance of potential enveloped viral contaminants, says Huffer. He also points out that barrier filters used for mitigation of viral contaminants are newer than standard viral reduction filters; however, they are especially designed to reduce the risk of a viral contamination event. “These are valuable in risk mitigation for gene and cell therapy products,” he says.

Huffer adds that new molecular methods, such as next generation sequencing (NGS), can be used to screen process materials going into a manufacturing process that do not have the option of viral clearance. “These new technologies reduce the risk of introducing a viral contaminant into a manufacturing process with limited or no opportunity for viral clearance,” he notes.

NGS can be a significant advantage with the possibilities to detect both known and unknown adventitious viruses, Jaber says, noting that characterized virus stocks and standard process steps will help validate the NGS platforms.

“Cell and gene therapy product manufacturing involves multiple steps, long culture, and processing times, and therefore may allow amplification of adventitious agents. Starting material and raw material qualification and testing [are] critical to ensure the safe manufacture of products. For example: implementing methods to remove or inactivate virus in media or components, development of media that do not require animal- or human-derived raw materials, and use of closed system to avoid contamination by environmental sources,” Jaber includes.

Meanwhile, traditional resin chromatography has been the mainstay of downstream processing for most biologics, Breton says, but with innovations in membrane chromatography, the industry has seen greater interest in these techniques. “For gene therapy products, we are starting to see monolith technologies utilized due to their defined flow path diameters, which are uniquely suited for larger products such as AAVs,” he states.

“Additional chromatographic techniques have been helpful in providing additional product purity and viral safety in the viral vector space,” says Schwartz. “For cell therapies, there are several advances that aid in setting an upstream viral barrier. These include virus retentive filters designed for media and the implementation of closed single use technologies,” he adds.

In discussing the unmet needs in the industry regarding viral clearance studies for cell and gene therapies, Schwartz asserts that there needs to be additional effort around the safety of cell therapies in particular. “Most risk analyses provide only barrier technologies and not true in-process viral clearance options. Having additional downstream tools that provide this clearance without impacting cell viability would change cell therapy processing,” he says. He further states that additional effort can also be made to develop faster and more effective viral clearance testing services to lower the bar for these new modalities to enter the clinic.

“Cell therapies do not typically have any downstream purification that could have the potential to remove or inactivate viruses,” Jaber also adds. Thus, viral clearance is not an applicable test method for these therapies, he states. “Therefore, animal- or human-derived raw materials/ancillary materials used in the production of cell therapy products should be critically reviewed, and if viral clearance is applicable in the production of such materials it should be applied,” he cautions.

Because it is currently difficult to remove viruses from cell or enveloped gene therapies without potentially damaging the product, finding a solution would greatly provide an unmet need for these products, says Huffer. Purification steps that provide an opportunity to selectively inactivate or remove a viral contaminant yet keep the biological product intact and activewould increase viral clearance opportunities for these products. “In the absence of new viral clearance steps for cell and enveloped vector gene therapies, new, sensitive molecular techniques that can identify viral contaminants in starting materials would add to increased viral safety,” he states.

Meanwhile, Breton points out that strong regulatory expectations for new biologic modalities in terms of viral clearance studies is the proverbial “elephant in the room” and that new science and new modalities are rapidly outpacing regulatory determinations. “This leads to feelings of uncertainty but, moreover, can potentially lead to inefficiency in bringing a much-needed product to market,” he states.

Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin

Feliza Mirasol is the science editor for 

When referring to this article, please cite it as F. Mirasol, “Viral Clearance Testing Increasingly Challenged by New Biologic Modalities,” 

Downstream Processing

New biologic modalities, such as cell and gene therapies, pose increasing difficulties in viral clearance testing methods.

Viral Clearance Testing Increasingly Challenged by New Biologic Modalities

The term “clean factory” was developed at the beginning of the 1970s with the birth of contamination control in the microelectronics industry, which introduced complex design manufacturing, defect control, and particle reduction. These demands grew beyond the cleanroom to utilities, equipment, and consumables. The late 1980s expanded contamination control beyond the manufacturing floor to the building and the parking lot.

Management of people, materials, processes, supplies, and utilities became a way of life for all types of cleanroom manufacturing, both industrial and healthcare. Additionally, in the early 1980s, the term “clean build” was initiated to promote controlled construction and reduce the risk of contamination from the design state until manufacturing.

Since then, decades of cleanroom technology brought about new tools and equipment, contained processes with isolators and mini-environments, increased contamination control standards and regulations, and increased customer demands for quality. The area concept established as part of the clean factory instituted protective barrier zones beginning with the process/product area and continued to the building exterior (1). The concept of defining seven zones for cleaning and sanitization worked well for years. The seven zones are:

Zone 1: Critical zone (equipment, process tools, and workstations that require the highest level of protocols)

Zone 3: Entrances to cleanrooms (personnel and materials)

Zone 4: Controlled environments (areas surrounding the cleanrooms)

Zone 6: Building entrances (visitors, employees, warehouse)

Today, the pharmaceutical industry faces many challenges: quality for customers, international competition, costs, and changes in regulations. This article will augment the original seven zones of the clean factory by adding specific modifications for personnel and facility. “Clean factory system” is a more inclusive term that can be used for these enhancements and updated cleaning protocols.

Most programs begin at the controlled environment to reduce contamination that enters the cleanroom. The clean factory systems approach, however, addresses contamination from the parking lot to the critical zone. This article will focus on personnel, cleaning, and facility design and will also discuss clean construction protocols and facility start-up. 

One of the key elements in the clean factory system is the selection of products for gowning and cleaning. The materials must support the contamination control program, reduce risk to quality in products, and meet the specifications for the class of cleanroom (i.e., particulate and if required, microbial levels). Additional gowning may be specific to product or process requirements. Other qualifiers, such as safety and electrostatic discharge (ESD), further define the selection process.

For gowning to be effective, the contamination from people must be addressed. Good sanitization and health habits are requirements of good manufacturing practices (GMP) (2). To help reduce particulate and microbial contamination, dedicated garments under cleanroom apparel are recommended. This type of garment replaces street clothing. Benefits to removing street clothing include:

Eliminates risk from “home” contamination (e.g., pets, food, transportation to work)

Use of a common fabric that is less particulating than street clothing

Use of a common fabric that is laundered in a cleanroom laundry

Specific design of the dedicated garment (e.g., long sleeves, proper closures).

 represents a compilation of data from numerous companies over the past 30 years and shows the reduction in particles in a gownroom (3). Clean street clothing offers very little particle reduction when compared to specific restricted clothing: no heavy linting fabrics, no shorts. Building suits, which are controlled with laundry and fabrics, offer some reduction. Cleanroom scrubs made of low-lint fabrics and laundered in a cleanroom offer the best particle control for the gownroom.

Cleanroom gowning must follow the standard operation procedure (SOP) or protocols. Gowning materials and methods must be reviewed to ensure that the materials meet the following standards:

Transfer of garments from warehouse to in-use storage

Storage of used garments, if not changed at each entrance

Life cycle of laundered gowns (number of washings allowed)

Repairs, stains, damaged garments replacement cycle

These are all critical considerations, and each zone in a plant should evaluate and document the type of gowning and materials that will support product quality and personnel protection.

The zonal concept does not address separation as recommended today. If a company manufactures multiple products, there are specific protocols for the changeover of the cleanrooms that must be performed to reduce cross-contamination. Companies should consider the risk and address specific changeover protocols for personnel, cleaning and sanitization, and facility design.

In a GMP facility, manufacturers are expected to prevent or mitigate contamination from particulates and micro-organisms that could impact the safety and quality of products. All tools, equipment, and supplies used for maintaining the cleanliness of a cleanroom or controlled environment must be constructed of non-shedding materials that can be easily maintained. The methods and techniques of cleaning and sanitization of cleanrooms and controlled environments should follow IEST RP-18 (4) or in-house validated methods and techniques based on particulate and microbial removal efficiency.

The cleaning materials must be compatible to the finish of the surfaces to be cleaned. This will prevent particulate generation during the cleaning process. The cleaning agents and disinfectants must ensure compatibility with the product, process, cleanroom surface finishes, and environment (waste disposal handling).

Water used in the dilution of disinfectants or cleaning solutions must be equal to the process/product water. Additionally, the tools, equipment, supplies, and solutions for cleaning and sanitization of areas outside of controlled and classified areas must never create a risk to these areas. The selection process for these areas must consider the potential risk of cross-contamination from improper or incompatible chemicals. Tools, equipment, and supplies could create sources of contamination if inadequately cleaned, as could the use of unfiltered water and re-use of mops and contaminated mop materials. Although these areas are not controlled environments or classified cleanrooms, the methods and techniques used for the base building must be adequate to clean the surfaces.

Companies should review airlock procedures to institute more frequent cleaning, and sanitization of surfaces in the production. Surfaces that are contacted frequently, such as door handles, equipment latches, bench/counter tops, and control panels should also have a higher frequency of cleaning.

The recommendations for each zone address potential features to control contamination and cross-contamination.

This area requires the highest level of protection (facility design), cleanliness, and personnel protocols. The requirements must be specific to the class of cleanroom, product, process, and customer or patient requirements for quality, regulations, and standards. This zone could be a specific area within a cleanroom or an entire critical cleanroom.

Gowning must follow the SOP for the class of cleanroom. Critical apparel generally include: coveralls, hood, mask, boots, gloves, and eye protection.

 Protocols are based on classification of the cleanroom, the levels of particulate and microbial control, and the surface finishes. Tools must be compatible with surfaces and cleaning solutions. Mops should be a continuous filament microfiber material, sterile or non-sterile.

Critical equipment cleaning will require wipes (sterile or non-sterile) and tools that meet the process requirements for particle count, compatibility with cleaning agent or disinfectant, and extractables.

Critical zones should have separate entrance and exit airlocks for personnel and materials.

The cleanrooms range in classification from ISO 1–8.

 The gowning for this zone follows specific protocols for the quality of the product, process, classification of the cleanroom, contamination levels (particulate, microbial, electrostatic), standards, regulations, and any customer specifications. In selecting the proper gowning, you must consider first the classification of the cleanroom as the prerequisite.

The protocols are based on classification of the cleanroom and the levels of particulate and microbial control and the surface finishes. Tools must be compatible with surfaces and cleaning solutions.

The entrances into cleanrooms should be through personnel airlocks (gownrooms), corridor airlocks, or main traffic corridors that are the same classification.

Before entering a cleanroom or controlled environment, proceed to a locker room and wash hands. If required by protocol, remove street clothing and don building suits, plant uniforms, or scrubs.

Garments can become contaminated if the gownroom is not adequately cleaned. Because of the traffic volume, these areas should be cleaned with a higher frequency than the manufacturing areas. This level of cleaning will reduce the risk of contamination to the garments, personnel, and the cleanroom itself.

If the gowning requires the removal of street clothing as well as street shoes, extensive locker room space, shoe storage, and garment storage is required. Gownrooms must be designed to support the population density, gowning requirements, storage of garments (if reused), and exiting requirements.

 Personnel should proceed to designated areas and store all personal items. Food items should not be contained in lockers that are adjacent to controlled corridors. Badges must be sanitized, and all personnel must wash hands. Some controlled environments have additional requirements, such as safety shoes, specific clothing (non-linting), no bare legs, no sleeveless tops, no jewelry, and no cosmetics.

Dedicated low lint microfiber mops, filtered cleaning solutions, and dedicated tools are required for these areas.

The surrounding environments will influence the cleanliness of the cleanroom. As people and materials are moved from Zone 5 to Zone 4, there is a risk of cross-contamination. It is important that separate airlocks are required for personnel and material entering or leaving controlled areas.

This area includes rest rooms, offices, cafeterias, lobbies, personal storage areas, and conference rooms.

Recommend hand sanitization stations throughout the building in public areas.

 Floors that are not maintained will create a risk of cross-contamination into the controlled environments. Cleaning should follow the protocols as outlined in Zone 6.

One of the biggest risks to the controlled environments from the base building is carpeted floors. Plants should consider the use of flooring materials that can be mopped daily.

 At the entrance, all personnel should step on a mat to remove heavy debris from their shoes and use a shoe cleaner if available.

Specific maintenance procedures should be established. All entrances must be cleaned at least once per shift to control the risk of contamination into the building. Entrance areas should be maintained free of visible debris and dirt and vacuumed, followed by wet cleaning. Warehouse areas should be free of all visible debris. Warehouse cleaning tools are dedicated to this area only.

Entrance areas should contain local area mats that withstand heavy foot traffic and can be cleaned multiple times per day. Shoe cleaners should be considered as an additional method of contamination reduction. Warehouse design must consist of an airlock for the transfer of items from the warehouse to the base building. An airlock will control the risk of contamination from the exterior during transfer of transported items.

The outside appearance creates the mind-set for all personnel and visitors.

 Clean clothing should be worn. If personnel will be working in controlled environments or classified cleanrooms, they should follow the protocols established for those areas.

Seasonal outdoor maintenance must be performed (e.g., clearing of leaves, snow and ice, debris). Loading docks must be checked weekly and cleaned to prevent food debris.

 The entrances to the building (warehouse, visitor lobby, and employees) should have airlocks that will act as a buffer from the outside world. Entrance, exit, and emergency doors must have proper seals. The control of the building exterior should be reviewed for types and location of plants, pest control, drainage of water, grading, and snow removal, for example. 

Clean build protocols should be considered to reduce the risk of contamination to the controlled environments and cleanrooms. The purpose is to establish and define the rules of entry, material transfer, construction activities, and personnel conduct. The specification delineates all procedures and requirements for all individuals gaining access to the areas and for entrance of all materials, equipment, and tools. As each phase of construction progresses, increasingly more stringent requirements are defined for personnel, material, equipment, tools, and cleaning. These requirements will provide appropriate levels of contamination control to the stage of construction and the intended designation for the area. All construction project personnel should follow the personnel guidelines as outlined for the clean factory system if transitioning from the exterior or through entrances.

Cleanrooms and controlled environments do not operate without careful consideration to their design, impact of contamination from the surrounding areas, personnel protocols, and specialized cleaning procedures. Cleanrooms and controlled environments must meet the specifications for particulate and, if required, microbial levels. These levels can only be achieved on a consistent basis by controlling the environments that surround these areas. SOPs are generally specific to an area and may not incorporate the entire facility system. The clean factory system concept, however, can be used as the foundation for analyzing gaps in contamination control for personnel, cleaning practices, and facility design, creating 360 degrees of cleanliness.

1. A. Dixon, “Chapter 11: Cleaning of Non-Product Contact Surfaces,” in 

3. A.Dixon, Data compilation from unpublished reports (2021).

IEST RP-18, Institute of Environmental Sciences and Technology Recommended Practice CC018.5, Cleanroom Cleaning and Sanitization: Operations and Monitoring Procedures

Anne Marie Dixon-Heathman, annemarie@cleanroommanagement.com, is principal of Cleanroom Management Associates, Inc.

When referring to this article, please cite it as A. Dixon-Heathman, “360 Degrees of Cleanliness,” 

Manufacturing

Help prevent contamination in a facility by zone.

360 Degrees of Cleanliness

The pharmaceutical regulatory landscape and pharmaceutical development have been reshaped by International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Harmonised Guidelines. ICH guidelines Q8–Q12 (1–5) and those in development such as ICH Q14 (6) have applied science, risk management, and quality systems to enhance process and product quality.

In 2011, FDA’s revised process validation (PV) guidance (7) extended ICH’s concepts to the pharmaceutical product lifecycle, an approach that is now being applied to cleaning validation and other areas of manufacturing, as well as to clinical trials (8).

This article proposes an independent structured assessment process that uses data to develop insights into product sustainability and risks. It focuses on the evaluation of intra- and inter-batch variability, and can be applied to both large- and small-molecule pharmaceuticals (9). This review process involves comprehensive process assessment (i.e., historical data review and the gathering of end-to-end manufacturing process data); extensive process monitoring and characterization exercises that enable lifecycle quality gaps to be addressed. The assessment process results in a heightened level of product and process knowledge, while substantially reducing the risk of potential failure.

Based on the 2011 FDA process validation guidance and relevant ICH guidances, this assessment method requires the use of analytical and interpretive methods based on standard statistical and data visualization tools. The approach allows results to be verified at any time, by both product owner and regulator, utilizing standard statistical tools. All data sets prepared, transposed, and analyzed should be subject to data verification procedures to assure data accuracy and integrity.

Documents required for the assessment include the following:

analytical test methods and test method validation

process performance qualification (PPQ) protocols and reports

The assessment uses current regulatory standards for validation (e.g., from FDA, the European Medicines Agency, Health Canada, and the World Health Organization). It also uses tools and methods such as cause-and-effect Ishikawa diagram, scientific rationale checklists, decision trees, as well as supporting analytical and statistical tools such as Minitab or JMP software.

The assessment focuses on analyzing the information and knowledge gained from the three stages of the process validation lifecycle: process design, process performance qualification, and continued process verification during commercial manufacturing. It is performed in the following two parts:

Part A involves a holistic regulatory compliance review solely focusing on the compliance with the lifecycle-associated lifecycle regulatory guidance including ICH Q8, Q9, and Q10, and future industry trends including ICH Q12, Q13 (10), Q14 and how they work in tandem.

Part B is a diagnostic study of the current process control (a seven-step product assessment process) that results in determining the adequacy of the current control strategy.

Part A. Compliance review: Adherence to FDA process validation and ICH guidelines

A comprehensive compliance review is designed to summarize and confirm compliance with the FDA process validation guidance and ICH and other emerging guidance, based on observation and the available data. The recent ICH Q12 document allows use of lifecycle guidelines for seamless post-approval change management. Adherence to lifecycle guidance therefore becomes more important for ongoing operational flexibility and compliance.

Stage 1, process design assessment, involves the following steps:

determining the quality target product profile and justification of critical quality attributes (CQAs) and critical process parameters (CPPs) as per ICH Q8

applying quality risk management tools as per ICH Q9

documenting a critical manufacturing equipment suitability assessment

determining degree and understanding variability

Stage 2, process performance qualification review covers aspects such as the following:

design considerations of a facility and qualification of utilities and equipment

validation of analytical methods per analytical quality by design (QbD) as per ICH Q 14

utilization of cumulative data from all relevant studies to establish the PPQ manufacturing conditions

number of batches and samples to provide sufficient statistical confidence

application of statistical process performance indicators

clear documented justification for manufacturing process to be in a state of control.

Stage 3, continued process verification of the product includes the following:

Continued monitoring and sampling of CPPs and quality attributes is at the level established during the process qualification stage until sufficient data are available.

An ongoing program is in place to collect and analyze product and process data, detecting unplanned departures from the process.

The data collection plan and statistical methods to determine process stability and capability are adequate in identifying variability in the process to signal potential process improvements.

Review of failures, non-conformances, and trends during commercial manufacturing are conducted.

Part B. Control strategy review: The seven-step legacy product assessment

The seven steps involved in Part B are as follows:

Variability is caused by contributing factors associated with materials, methods, machine, measurement, manpower, and mother nature (11). In manufacturing, this relates to attributes attributes of material, formulation, and associated processing parameters including scale-up factors, processing equipment, test method, personnel, and facility/utilities (

). The overall CQA variability is a function of individual component variability.

A cause-and-effect technique is utilized to identify underlying factors that could have an impact on CQA that would affect patients (i.e., label claim CQAs). The assessment includes factors and attributes of equipment, personnel, and facility/utilities as show in 

Upon identifying the risk factors through cause-and-effect techniques, a risk-rating decision tree (

) is used to analyze the process upon risk assessment. The criticality assignment will be commensurate with the product/formulation characteristics. In addition to any product-specific CQAs that would affect the patient, the review should—at a minimum—assess the impact of variability factors on CQAs. Risk assessment must cover all attributes.

The decision tree with a data-driven semi-quantitatively risk-rating system (

) is used to prioritize the unexamined influencing factors in yellow (

) identified for each element. Additional review is warranted when the rating value is higher.

The decision tree-based assessment (Step 2) determines whether the established current control strategy for each attribute is supported by the cumulative documented data from relevant Stage 1 studies (e.g., design of experiments, development batches). The control strategies established to control and mitigate the identified risk factors are then reviewed to determine the missing parts.

This gap assessment also evaluates whether the established current control strategies are derived based on objective information/rationale and data from original and subsequent process design studies. The need for establishing additional controls, including manufacturing process controls, is determined at the end of the gap assessment.

The gap assessment also determines whether the established current control strategy is supported by the cumulative data from all relevant studies (e.g., designed experiments, development batches, and PPQ batches). The control strategies established to control and mitigate the identified risk factors are reviewed to arrive at the conclusions. The residual risks and the need for establishing additional controls, including manufacturing process controls, are then identified to initiate a heightened sampling and testing Stage 3a plan for the product.

Lifecycle review and Stage 3a monitoring batch execution (Step 4)

The lifecycle review should determine, based on available lifecycle data, whether the current process is operating consistently within the originally established control strategy. The control strategy gap assessment step also may identify the need for additional controls or identify gaps in supporting data. The lifecycle review will determine if any commercial product data supports the identified gaps.

Data from all three stages of the process validation lifecycle (e.g., design of experiments, manufacturing trials, statistical analysis, annual product review, and commercial batch data) may enable the development of a risk-based control strategy that would suitably minimize sources of variability. Data from previous credible experience with sufficiently similar products and processes may also be utilized to support the control strategy.

Data gaps identified at Stage 1 QbD, Stage 2 equipment/facility/utility qualification and PPQ, and Stage 3 continued process verification require that a sampling and testing plan be developed under a Stage 3a protocol to further enable prediction with adequate statistically significant data sets.

The type of Stage 3a sampling/testing plan developed is based on the product/process gaps that have been identified. The Stage 3a study focuses on processes that have not been characterized, or for which variability has not been determined. Extensive data must be collected and tested and a monitoring protocol executed by the technical operations team responsible for Stage 1 lifecycle of the product. The Stage 3a lifecycle monitoring data that are collected supports the identified gaps or enables closure of existing Stage 1 data gaps for the product.

Data-driven, scientifically sound Stage 3a monitoring enables the following:

continual enhancement of the product control strategy

collection and evaluation of information and data, such that sources of variability can be detected, as recommended by FDA process validation guidance

closure of the characterization gaps that were not addressed during early product development and to meet QbD standards, per ICH Q8 (R2)

determination of intra- and inter-batch variability as recommended by FDA process validation guidance

development of a baseline for Stage 3b monitoring as recommended by FDA’s process validation guidance.

ICH Q10 recommends management of product and process knowledge from development through commercialization and until discontinuation. The monitoring batches add to the body of knowledge on the product. FDA’s process validation guidelines allow for use of credible experience with sufficiently similar products; hence a bracketing approach for monitoring may be applied and conclusions can be used for control strategy enhancement of similar products.

The Stage 3a data collection protocol (12) developed after gap assessment enables in-depth statistical assessment of the product. The Stage 3a assessment will also provide a baseline for continual Stage 3b program for the product. The approach supports the organization’s ability to make science- and risk-based continual process improvement decisions for the product as well as to identify areas where QbD development should focus for similar manufacturing processes/products. The Stage 3a monitoring measures are developed in line with upcoming revisions to ICH Q2/Q14 and the proposed 

 General Chapter <1220> framework for analytical QbD which requires identification of adverse trends, allowing proactive measures and facilitation of continued improvements and change control through continued monitoring.

The extensive data collection step includes batch data from development, process performance qualification, and commercial manufacturing. The new database will include potential variables that can impact the CQAs. This will include, but will not be limited to, materials, upstream and downstream process parameters, equipment parameters, hold times, operational variables, operation/analytical personnel, instrumentation, documented weights/time, facility/utility parameters, and incidents. This step will involve extensive data set collection, data set review, and data preparation for the subsequent statistical analysis.

As a result, the database must be developed so that it maximizes the utilization of existing product/process data for multivariate statistical analysis such as correlations. The database is created for variables that can impact CQAs. The database is utilized as a starting point for Stage 3b trending and ongoing signal detection of products that are determined to be robust post assessment.

The data collected from Stage 3a heightened sampling and the historical batches are statistically analyzed to identify patterns and insights into the impact of potential sources of variability on the CQAs. Statistical software tools such as Minitab and JMP may be utilized. The analysis determines the adequacy and suitability of the collected data sets to support the current control strategy. The analysis first attempts to determine whether the original control strategy developed adequately meets the current standards.

A structured assessment process can determine compliance to lifecycle process-validation requirements for biopharmaceuticals.

BioPharm International, July 2021 Issue

Defining a Holistic Approach to mAb Purification Processes

Viral Clearance Testing Increasingly Challenged by New Biologic Modalities

360 Degrees of Cleanliness

Assessing Manufacturing Process Robustness

Preparing for the Unexpected: E&L Studies in Biopharma

Track-and-Trace Progress Benefits Supply-Chain Security Efforts

Balancing Pressing Priorities

White House Promotes US Drug Manufacturing to Secure Access to Vital Medicines

Avoiding Delays on the Road to Normalcy

The Value of FDA 483s and Warning Letters