The personalized medicines era is shifting the focus of pharmaceutical development and manufacturing activities from small molecules to biologic drug products. The formulation of these complex macromolecules, however, is not as straightforward as their small-molecule counterparts. The high patient doses required for biologic therapeutics, for example, present significant drug-delivery challenges, according to Johann Stasny, director, Global Sales Development, Bioavailability Enhancement & Custom API, Merck Millipore KGaA, Darmstadt, Germany. “On average, a dose of 0.1 mg to 3 mg per kg of patient body weight is required,” Stasny notes. Because of their nature, biopharmaceuticals are not easy to deliver via the oral route, explains Kate Hudson-Farmer, market insight strategist, Drug Delivery, Cambridge Consultants. A large proportion of these drugs are currently injected using intravenous infusion in the hospital, or increasingly in the home, she says.


	Most biologic products for parenteral administration are freeze-dried in vials to help maintain drug stability and increase shelf life. But interest is growing in the development of liquid formulations in prefilled syringes and autoinjectors, which offer convenience and ease of administration in a home setting. Stasny observes a shift for certain biologics-especially those indicated for chronic conditions such as inflammatory diseases-toward prefilled syringes for subcutaneous administration. “This delivery method has better patient compliance and efficiency in cases where a high concentration of the drug is required due to the low dosage volume that can be administered,” he says.

	“The trend in self-administration continues to grow,” Hudson-Farmer notes. Claus Feussner, PhD, senior vice-president, Development Service, Vetter, attributes it to the expanding home healthcare market, which he says is a direct reflection of a constantly aging population, particularly in the more established countries. And as a means to control costs and avoid expensive therapies in either a hospital or a doctor’s office, healthcare authorities are putting pressure on the industry to develop medicines that patients can self-administer in a private setting, according to Feussner.

	Hudson-Farmer, nonetheless, points out that self-administration has its challenges. Patients require training and education to ensure that these drug-delivery systems are used correctly. “But this is not the only problem,” she asserts, “because self-injection, whichever way you look at it, is not a pleasant thing to have to do, and adherence and compliance to such drugs is often a problem.”

	In addition, drug companies are faced with the challenge of having to deliver larger volumes of biologic formulations, often beyond the capacity allowed by a syringe or an autoinjector. “This is in part due to the trend towards less frequent injection and in part, to drug formulation issues,” says Hudson-Farmer. Patch pumps or on-body injectors that can deliver the drugs in a subcutaneous manner are being developed, she adds. “There have been some interesting advances in this area including the pre-timed, worn-on-body injector that Amgen launched for its Neulasta drug using Omnipod’s on-body injector,” Hudson-Farmer highlights.


	Andy Fry, founder of Team Consulting, notes that wearable devices, which provide an alternative to prefilled syringes and autoinjectors, are attracting a great deal of interest. “A wearable device allows drug delivery over a significantly longer period than the typical 10- to 20-second window associated with an autoinjector. Because of this feature, a wearable device can enable large-volume doses, or high-viscosity formulations, or even larger-volume payloads at high viscosity, to be delivered at acceptable levels of patient discomfort,” he explains. “There has also been renewed interest in all-in-one dual-chamber devices, which aim to take the fiddle and complication out of reconstitution of lyophilized products for self-injection while avoiding the necessity of cold-chain transportation and storage.”


	Graham Reynolds, vice-president of marketing and communications, Delivery Systems, West Pharmaceutical Services, sees an increased need for innovative and effective drug packaging and delivery of biopharmaceuticals. “Additionally, as biologics begin to come off patent, demand for delivery systems that can safely contain and deliver biosimilars should continue to rise, especially as easy-to-use delivery systems can help differentiate drug products,” he says.

	The industry also continues to seek alternatives to glass, such as polymer-based systems, for drug packaging and delivery systems, according to Reynolds. “This is particularly true for biologics, which can be sensitive to their containers,” he observes. “Glass-related risks, such as delamination, breakage, and chemical incompatibility with the drug product do not exist with the newer polymer offerings. These platforms offer a range of options for dose volume and injection time and are designed to help improve patient compliance and outcomes, while meeting the challenges of injectable drug products.”

	Biologics are sensitive drug products, explains Reynolds, emphasizing the need for ultra-clean packaging components to ensure that there is a low risk of contamination or particulate. “Manufacturers and regulators are demanding that drug containment and delivery systems have the highest levels of quality possible to maintain the purity of these sensitive medicines,” he asserts.


	According to Stasny, a holistic approach is required to better understand the full process by which the formulation of a biologic drug product is implemented. “Biologic processing, in general, is evolving to a more efficient platform,” he says. “High-titer bioreactor production, higher capacity chromatographic systems, and continuous processing strategies will create additional stresses on biologic products during processing to achieve the final formulation.”

	Stasny explains that higher concentrations of biologics throughout the purification process and into the final formulation create greater chances of intermolecular interaction, thereby leading to higher potential for aggregation. “Once the final formulation has been optimized and assessed, challenges such as viscosity derived from high-concentration systems have equivalent impact on the ability to process, formulate, and deliver the biologic drug,” he continues. “Formulation design through appropriate selection of excipients and process chemistry may be employed to mitigate both aggregation and viscosity challenges both in the final dosage form and upstream into the process to improve yield and efficiency. Because aggregates have a considerable impact on immunogenicity, minimization of aggregation potential through appropriate process and formulation design will provide an additional benefit.”

	In addition, the qualification of the excipients and process chemicals used are important, says Stasny, not only to meet regulatory requirements, but also because the quality attributes can reduce oxidative contaminants. “Identification and control of levels of reducing sugars, peroxides, aldehydes, elemental impurities, and other detrimental contaminants in both process chemicals and excipients will reduce oxidative potential, leading to improved stability,” says Stasny.

	In the following roundtable discussion, experts spoke to 

 about the key considerations in the development of a drug-delivery device for a biologic drug, the importance of human factors engineering, the advantages of prefilled syringes, and the challenges involved in the manufacture of these devices. Participants were: Hudson-Farmer from Cambridge Consultants; Reynolds from West Pharmaceutical Services; Feussner from Vetter; and Fry and Julian Dixon, director of human factors, both from Team Consulting.

: How do you develop a successful drug-delivery device for a biologic drug?

: Success is a combination of many factors. Most of the formulation issues do not affect the development of conventional devices like auto-injectors, as stability is governed by compatibility with the primary pack (usually a prefilled syringe) and not the rest of the device. However, with the move toward self-administration, resolving formulation issues such as stability without the appropriate consideration of usability challenges success. Lyophilization, for example, is a good way to stabilize a drug, but it increases preparation complexity for the patient due to the need to reconstitute the drug prior to injection.

	Self-injection opens up a variety of delivery opportunities compared with conventional oral delivery of drugs. Ensuring there is some degree of flexibility for the patients using these devices, and the healthcare workers assisting and advising the patients, is key. There will be distinct needs for certain patient groups that vary from therapeutic area to therapeutic area. Designing the device of choice for a rheumatoid arthritis patient, for example, may differ significantly to a patient who needs to use it as a rescue device for allergies.

	There are also differences in needs within a therapeutic area. Some may want a simple device that works quickly, and they can forget about the condition they have as soon as injection is completed. Others may be more interested in understanding their disease and managing it closely, and could benefit from more interactive features, such as those offered from connected health add-ons.

	Product differentiation using the device element of the therapy can give the pharmaceutical company a competitive edge, but there has to be a balance in terms of the increased cost and complexity of sophisticated device offerings. The device can present technical formulation challenges besides the challenges related to the optimization of the device configuration, such as the need for new primary packaging materials. Keeping certain aspects consistent, such as primary packaging, is important so that new regulatory demands are not placed on the drug. A device platform that could be altered to improve patient experience, without having to take the whole device through trials for every alteration, would be very appealing.

: Easy-to-use dual chamber reconstitution systems can side-step stability issues with a configuration that is relatively easy to use. Protein aggregation is usually associated with silicone oil used to reduce stopper friction; therefore, steps to reduce or even eliminate silicone oil are worth exploring. These steps may include much closer control of the siliconization process, using low-friction stoppers, or cyclic polyolefin syringes that require no silicone lubricant. Wearable devices have the capacity to manage both high volumes and high viscosities, so a wearable, dual-chamber device may well have the potential to tick several boxes at once.

	When it comes to developing a successful drug-delivery device for a biologic drug, it is always better to start early. All too often, device developers are approached when the biopharmaceutical company has already made all the formulation decisions and presents a ‘fait accompli’ formulation with little or no room to explore potentially worthwhile options, which might have existed had some dialogue taken place earlier. Two major issues that are sometimes left too late are device usability and manufacturability. These issues must be part of the design thinking and the development process from the earliest point possible.

: Can you elaborate on the importance of human factors engineering (HFE)?

: The importance of HFE has increased dramatically over the past few years, and its impact on human safety has resulted in greater attention from the regulators, particularly FDA. The concern is increased further by the move towards self-administration, which increases the risk of usage error. FDA has introduced guidance (1) that calls for HFE to be integrated into combination product development as part of the risk-management process. A combination of analytical methods and observational studies is recommended to identify causes of error and assess and propose mitigations. A final validation study is then conducted to show that the usability has been optimized.

	The use of HFE, although still in its early days, can potentially reduce usage error and allow for safer products. A possibly more exciting situation will be to use HFE to support better patient engagement and address the wider issue of non-adherence. In this case, the issue moves from making devices that people are able to use to making devices that people want to use.

: HFE is important in this arena for two different reasons. Firstly, it is a regulatory requirement to demonstrate that HFE has been considered throughout development. The regulatory bodies, particularly FDA, are correctly stressing that drug-delivery devices need to be safe and effective in the hands of their users. It is important, therefore, that an appropriate HFE plan has been followed. Failure to do so, even if the drug-delivery device is well designed, represents a significant regulatory risk, and the product may not be approved as a result. 

	Secondly, a balanced consideration of HFE throughout development will ensure that the drug-delivery system is as good as it can be. The drug-delivery field has been late to embrace user-centric and human factors principles. There is now a general agreement on their importance. The best way to develop drug-delivery devices is to have a well-integrated team of technologists, formulators, engineers, designers, and human factors experts. Another key takeaway is that HFE is important because failure to consider its inputs to design and development will result in a suboptimal product.

: Do you see an increasing demand for prefilled syringes?

: The drug-development model today is far more intricate compared with some years ago, created by factors that include a higher complexity of new compounds in development. Companies hoping to succeed must make innovation central to their drug-development business approach, which includes looking outside traditional methods of drug delivery in clinical phases.

	The global prefilled-syringe market is expected to show continued positive growth due to rapidly increasing development of biologic drugs. There is also a rising demand for more user-friendly administration, starting in the clinical phase and continuing in the commercialization of a drug. With more biologics competing in the same therapeutic space, launching a drug product in a syringe can help set it apart from the competition.

Prefilled syringes remain a container of choice for many injectable drugs, especially for biologics, and there’s no indication that utilization will decline anytime soon. Because the prefilled syringe delivery system has so many formats using the same primary container, delivery can be customized based on need. For example, home administration is often best suited for autoinjectors, which can provide fully automated needle insertion, dose delivery, and needle retraction. For hospital-based administration, needlestick prevention systems can be essential to meet the requirements of legislation on safety and protect healthcare workers. In all cases, prefilled syringes are designed to meet the needs of not only the pharma/biotech companies, but also the patient.

: What are the advantages of prefilled syringes for delivering biologic formulations compared to other injection technologies?

: From a patient standpoint, prefilled syringes facilitate accurate, pre-measured doses, hence, eliminating the risk of over- or under-medication. Another advantage is that prefilled syringes in autoinjector formats allow patients to administer the drug from home, as opposed to visiting a healthcare provider, which reduces the amount of time spent in transit and eliminates a burden for patients with weakened conditions.

	Accessories such as extended finger flanges, needle shield removal systems, or ergonomic plunger rods can be valuable in addressing patient needs and can accommodate biologic drugs with higher viscosities or higher dose volumes. These advancements continue to position prefilled syringes as a more viable option for a wider range of patients.

: The key to the rapidly changing healthcare market is offering solutions for the need of patient-friendly systems. Instead of undertaking several preparation steps prior to administration, the prefilled syringe, also known as an all-in-one system, provides a number of advantages in the clinical stage as well as in the day-to-day operations of a product already launched. These advantages include:

		Improved trial appeal, making it easier to recruit medical clinics for clinical trials that use prefilled syringes instead of traditional vials

		Greater patient compliance and consistency resulting from the user-friendly nature of prefilled syringes

		Precision single-unit dosing, which better meets the requirements of today’s more complex compounds

		The avoidance of overfilling, which reduces loss and thus saves valuable API as compared to vials.

What are the challenges associated with prefilled syringes?

: Creating easy-to-apply systems often means a more complicated production process at the outset. A syringe system is a complicated instrument, consisting of a number of individual components. The correct conjunction and interaction of each module is decisive. For example, consider that administering a drug using a syringe requires a plunger rod that operates in concert with the other components, especially the glass barrel itself. To achieve this, a small coat of silicone is applied at the inner surface of the glass barrel. Finding the correct amount of silicone to enable the correct movement of the plunger rod, while avoiding any form of interaction between the silicone and the drug substance coming into contact with each other, is a significant challenge.

	Also, because the glass barrel of a syringe by nature is more complex than the glass barrel of a vial, there exists a higher risk for mistakes or damage to the barrel such as glass scratches, or even glass breakage. This is why all the partners in the supply chain have been focused individually and collectively on this issue for a number of years already. All players, from the glass manufacturers and the prefilled syringes manufacturers to the bio/pharmaceutical companies and their CDMOs, are striving to achieve optimal results for the benefit of the patient.

: Biologic molecules tend to be sensitive to products commonly found in traditional glass prefillable syringe systems, such as metal ions and silicone oil, substances that may affect the drug product’s efficacy and a delivery system’s performance. On a macro level, one of the biggest challenges is understanding the interactions between all elements of the drug-delivery system. This extends beyond just the drug, container, and delivery device, but also takes into consideration the most important part of the equation-the patient. Understanding all key elements of a drug-delivery system provides the cornerstone that enables this system to achieve the goals of encouraging adherence in the home-care environment. Drug manufacturers should seek a partner that can apply proprietary technologies, manufacturing excellence, and patient understanding to their drug products and the products’ packaging, delivery, and administration systems.

: What testing do you have to carry out to ensure sterility and stability of the formulation as well as compatibility between the prefilled syringe and drug?

: It is important to implement early testing for extractables and leachables. Complex chemicals that may be present in drug container and component materials can potentially interact with and affect the efficacy of the injectable drug products. Extractables migrate from packaging or other components and are found under exaggerated conditions, while leachables migrate from packaging or other components into drug products under normal conditions.

	It is crucial to detail all of the materials that come into direct contact with a drug product during every phase to reduce the risk of contamination. Extractables and leachables testing is recommended even if containers or components meet compendial suitability tests, and should be carried out as part of the qualification for the container and its components.

	Such testing includes liquid chromatography/mass spectrometry (LC–MS), gas chromatography/mass spectrometry (GC–MS), inductively coupled plasma (ICP), and ion chromatography (IC). The results will be a materials profile that offers a clear picture of components that come into direct contact with the product. Early extractable and leachable testing offers the opportunity to improve understanding of compatibility while reducing risk and expediting total development and launch timelines.

: For sterile injectables, there is a standard set of analytical tests needed to prove the sterility of the product for formal market release. It starts with quality control of the incoming components. For the drug filling production step, environmental monitoring of the surroundings and in process controls of the drug solution are usually carried out, including bioburden during filtration steps, sterility testing, and bacterial endotoxin testing. Furthermore, syringes are tested for integrity of closure through a number of qualified methods.

	The analytics used to assess stability of the drug are specific to the type of compound itself. Testing can be split into the standard chemical tests (e.g., pH, osmolality appearance, and residual moisture) and compound-specific characterization tests.

	Drug-stability testing is carried out to test for tolerance to syringe contact surface substances, such as silicone. The amount of silicone is tested using Fourier transform infrared spectroscopy or atomic absorption spectroscopy. In addition, the distribution of the silicone in the syringe can also be examined.

	Beyond silicone contact sensitivity, protein-based compounds can also be sensitive to a wide range of other factors, which include reactions to uncoated rubber or to the glue used to fix a staked needle. A number of analytics can be carried out to look for possible degradation products and the generation of denatured proteins.

Draft Guidance for Industry and Food and Drug Administration Staff, Applying Human Factors and Usability Engineering to Optimize Medical Device Design

When referring to this article, please cite it as A. Siew, "Drug-Delivery Systems for Biopharmaceuticals," 

Articles

Cover Story

Industry experts spoke to BioPharm International about the key considerations in the development of a drug-delivery device for a biologic drug, the importance of human factors engineering, the advantages of prefilled syringes, and the challenges in the manufacture of these devices.

Drug Delivery Systems for Biopharmaceuticals

There are multiple variables to consider in the reduction of bioburden, including the evaluation of microbes in a facility, in the manufacturing process, and in the materials used and produced in the process stream. Kathleen Souza, senior microbiologist in the Department of Virology and Microbiological Sciences at EMD Millipore, and Nick Hutchinson, market development manager, pharma and biotech at Parker domnick hunter, discuss the use of a combination of orthogonal approaches in the assessment of bioburden.
	
	Also included are comments from Marc Mittelman, PhD, senior managing scientist, Exponent, Inc. and Vince Anicetti, executive director of quality, Coherus Biosciences. Mittelman and Anicetti are co-chairs of the Parenteral Drug Association task force that developed the bioburden and biofilm technical report titled, 

PDA Technical Report No. 69, (TR 69) Bioburden and Biofilm Management in Pharmaceutical Manufacturing Operations

: What type of planning is necessary to target all aspects of bioburden reduction?

 The most important consideration in bioburden control is staff training at all levels within an organization. The planning process needs to involve representatives from QA/QC, manufacturing, and facilities engineering; an effective communication strategy needs to be developed to ensure that any bioburden excursions are immediately addressed. Understanding potential sources of contamination for a given facility and manufacturing process is key to planning for design, preventative maintenance, bioburden monitoring, and remedial actions. Effective bioburden control plans involve development of detailed, written standard operating procedures (SOPs) that address both preventative and remedial actions at each unit operation.

: A suitable risk assessment tool should be used to assess the likelihood of bioburden becoming problematic. Process maps showing process inputs can be constructed and the risk of these contributing to bioburden loads assessed. Chromatography resins, for example, are not typically provided sterile or capable of being sterilized, so they are a potential source of contaminations unless cleaned, sanitized, and stored correctly. Failure mode effect analysis (FMEA) and fishbone diagrams are useful tools to use.

: Planning the design of a comprehensive bioburden reduction strategy should start with risk assessments for each of the entry routes. The identified risks and severity levels inform planning of raw material selection, facility design, process elements and conditions, cleaning and sanitization requirements, establishment of alert/action levels, selection of monitoring and detection methods, operator training requirements, and quality system oversight.

: What procedure is used to identify the various points in which bioburden can be introduced? Is the identification process performed for each variety of biologic product?

: The creation of a process map will depend on each process. Indeed, some processes, like a cell therapy process, will need to be performed in a sterile manner, so no level of bioburden would be acceptable. My view is that if a good risk assessment has been performed on one variety of biologic, then the framework would be suitable for a second type with the relevant adaptations.

 Identification of potential bioburden entry points begins with effective communications between QA/QC, manufacturing, and facilities engineering staff. FDA guidance documents, PDA TRs, and the peer-reviewed literature all provide descriptions of potential point-sources of contamination. Bioburden may be associated with personnel, incoming raw materials (excipients, APIs, filters, etc.), compendial and non-compendial waters, and ambient air and associated airborne detritus. The potential point-sources for contamination are often common among different products, but the nature of the bioburden is influenced by the product. For example, mammalian cell lines or formulation constituents may require routine monitoring for mycoplasma or viruses. Open process operations are more susceptible to contamination than are closed processes.

: Bioburden may enter a manufacturing process or product through multiple routes, including cell banks, raw materials, utilities, equipment, the facility, or personnel. A risk assessment of each entry route using tools such as an FMEA will determine the risk levels and help target the highest risk operations or materials in the development of a comprehensive strategy. During a risk analysis, the process or environment is assessed for potential types of contamination. If components or equipment are different for processes, such as microbial fermentation and mammalian culture, a separate risk analysis is performed for each process.

: Does operating personnel need specific training on bioburden reduction?

 The manufacture of biologics requires operators trained in aseptic techniques, with specific training for relevant unit operations and cleanroom classification. Operators in the critical final fill area require training in practices that minimize product contact. Operators are trained on appropriate gowning procedures, required hygiene practices, and self-reporting of personnel conditions that could contribute to high levels of bioburden or potentially pathogenic contamination. Finally, operator training should stress compliant behaviors and commitment to established procedures.

: Training of personnel will be very important and will vary depending on roles and processes. In my experience, this is typically covered in GMP training, for example, gowning procedures, good aseptic practices, and specific operating procedures for operations, such as taking samples.

 All staff should be aware of the importance of bioburden control in both unit operations and in the finished product. Understanding the basics of microbial growth and routes of contamination is key to effective management independent of the products manufactured. Bioburden problems often result from a lack of understanding of personnel actions that result in a process or product contamination event.

: How do endemic bacteria affect the type of filter needed for filtration?

 While bacteria (and fungi) may be present in all in-process formulations, viruses and mycoplasma are typically only encountered in formulations containing serum or animal, plant, or insect cell lines. The selection of specific microporous membrane filters or ultrafilters for a given product should also be based upon experience with the formulation process and the required bioburden reduction at each step. Considerations such as membrane porosity, charge, or protein binding may also influence the selection process.

: Undoubtedly, understanding the organisms endemic within the facility plays a role. I have described previously the impact of mycoplasma species on cell cultures. There are a variety of approaches to controlling these organisms. I advocate the use of 0.1-micron rated filters that have been tested to demonstrate their effectiveness at removing these organisms for cell culture media filtration.

	Despite normal flow filtration being an established operation within the industry, we continue to see some relatively poor practices being performed. Confusion remains amongst bioproducers who believe that, if they are using a sterilizing-grade filter, they will automatically get a sterile filtrate. This is not necessarily true, and I think this can lull people into a false sense of security. How a filter is challenged and used in operation is extremely important.

: Biologics may be at risk for contamination from fungus, bacteria, mycoplasma, and virus in the bioreactor depending on raw material source. Risk of mycoplasma contamination in raw materials may warrant the use of a 0.1 um-rated filter, while risk of virus contamination may warrant the use of an upstream virus barrier filter. Virus nanofiltration is typically implemented downstream for removal of adventitious viruses from raw materials or endogenous retroviruses expressed by cells.

: Is there a specific method or process that you find to be the most reliable to ensure maximum bioburden reduction of biologics?

: A comprehensive approach-combining bioburden removal methods, process design elements, operator training, cleaning and sterilization methods, process monitoring, and quality oversight-is essential. A combination of orthogonal methods is most effective for minimizing bioburden during production. Culture media treatment by high-temperature–short-time (HTST) or UV-C will reduce microbial load. Filtration may be used for cell-culture media or buffers. Chromatographic steps, low pH, and chemical inactivation provide virus reduction during processing. Virus and sterile filtration prior to final fill ensure maximum bioburden reduction. This comprehensive approach will lead to greater assurance of patient safety.

Mittelman and Anicetti (PDA task force): 

There is no single process that will ensure effective bioburden control in a biologics manufacturing process. Point-source recognition, then employing effective controls at each unit operation, is key to effective bioburden management. Effective bioburden reduction begins with raw material and supplier controls. The PDA TR repeatedly notes that personnel are the most important source of bioburden--either through direct contamination or improper application of bioburden control practices. Ensuring that compendial and non-compendial waters that are used in the manufacturing process are maintained under good microbiological control is important: water is typically the single-largest raw material used and [one of] the most common sources of bacterial contaminants.

: Bioburden considerations will vary even if the same process is being performed in different ways. For example, when processing a small number of batches for clinical trials in multi-product facilities, there is a tendency to have long intermediate product hold times in order to account for uncertainties in process timings, equipment availability, and set-up times. If this is the case, then in-process bioburden filtrations and aseptic techniques become more important. The batch might be being processed over many weeks. As the volumes of intermediate product pools are typically small, they can be moved into cold rooms between processing steps to prevent microbial growth. At large scale, when campaigns are being manufactured to supply the market, it is more likely campaigns of a single-product are being run at a higher run rate in order to achieve operation efficiency. In these cases, the hold times are typically shorter, but there is less of an opportunity to reduce the temperature of intermediate product pools. As such, the approach that might be adopted for bioburden control will vary.

 When referring to this article, please cite it as R. Hernandez, "Selecting a Comprehensive Bioburden Reduction Plan," 

Downstream Processing

Process maps and risk assessments are among the valuable tools operators can apply to reduce the risk of microbial contamination.

Selecting a Comprehensive Bioburden Reduction Plan

Whether preparing GMP production or non-production master cell banks (MCB) and working cell banks (WCB), end-of-production (EOP) cell banks, or research and development (R&D) cell banks, mastering the art of cell bank (CB) production requires specialized expertise; optimal equipment and environment; appropriate quality controls; constant monitoring; close communication; and continual troubleshooting. Further, the process for characterizing these cell lines can be extensive, requiring an array of testing options for specific scenarios. From adventitious agent testing to identity and genetic stability testing, it is important to know what options are available, when is the best time to perform these tests, and how testing will impact the overall project schedule and outcome.


	MCB is defined as an aliquot of a single pool of cells that generally has been prepared from a selected clone under defined conditions, dispensed into multiple containers, and stored under defined conditions. A MCB typically originates from a research cell bank that is the “end point” of cell-line development. A WCB is prepared from a homogenous cell suspension obtained from culturing the MCB under defined culture conditions.

	Cell banks are typically classified via historical progression: R&D CBs, MCB, WCB, and EOP CBs. Another commonly used terminology reflects potential usage of a bank as well as its GMP status: non-GMP banks (or R&D banks), GMP non-production banks (e.g., bioassay banks), and GMP production banks.

	Generally, in GMP facilities, non-GMP (R&D) cell banks are prepared under the same general rules and conditions as GMP banks; however, strict adherence to GMP is not required. GMP non-production banks are typically produced in non-campaign mode, meaning that more than one bank can be prepared simultaneously in the same laboratory. Under these circumstances, appropriate cleaning and changeover procedures must be used to avoid cell line cross-contamination. GMP non-production banks are often used as indicator cells lines in cell-based potency assays.

	Recently, there has been an increase in demand for ready-to-use bioassay banks (RTU). The purpose of such banks is to use cells straight from the vial in the bioassay, thereby eliminating the cell expansion step and saving time in the process. RTU banks are usually large in size (400-1000 plus vials) and have a significant number of cells per vial. High cell viability (>80% or even >90%) is a common expectation, therefore, pilot studies are highly advised due to potential viability issues when scaling up from a small research bank to a bank of significant size.

	GMP production banks are mostly produced in campaign mode, meaning that only one cell line can be grown in a cell-banking suite at a time. Cells from a GMP production bank are used to make a product. The viability expectation for GMP production banks is typically >70%.

Cell bank biosafety testing: Risk assessment and regulatory expectations


	To ensure the purity and safety of the cell bank, it’s necessary to design and implement a comprehensive testing program based on a thorough, accurate risk assessment. As part of this assessment, it’s important to consider the type of cell bank that has been prepared, as the rationale for testing differs depending on the type of cell bank that is produced. For example, the testing program for a non-production cell bank (cells used for analytical assays) or for seed cells that are expanded to create a MCB is typically much more abbreviated than the testing program implemented for a production cell bank.

	Testing of all cell banks should include testing for mycoplasma and sterility, which should be performed prior to bringing the cells into a clean facility. Sterility testing ensures that the cells are free from bacteria, fungi, and mold, which can be introduced through contaminated cell lines, the use of contaminated animal- or plant-derived raw materials, operators, or contaminated facilities. Sterility testing is performed as per compendial methods. It’s important that bacteriostasis and fungistasis testing is performed prior to sterility testing to assess the sample matrix for inhibition.

	Similarly, mycoplasma are a common cell-culture contaminant, often sourced from the cells themselves, additives, or laboratory personnel. Mycoplasma contamination can not only lead to changes in the metabolism of cells, which ultimately affect product yield and quality but are also associated with human disease. Therefore, it’s important to ensure the cells test negative for mycoplasma. Standard mycoplasma testing is performed as per compendial methods and mycoplasmastasis testing is ideally performed prior to routine testing in order to assess the sample matrix for inhibition. Given that historical mycoplasma testing takes four weeks to complete, rapid methods have been developed that exhibit comparable sensitivities but that take only days to perform. Consequently, many companies are considering these methods as a viable alternative to the compendial 28-day assay.

	With respect to non-production seed cells, mycoplasma and sterility are typically the only biosafety tests performed. For non-production cells intended for use in analytical testing, additional characterization testing should focus on the performance of the cells in the particular bioassay for which they are intended.

	The regulatory expectation for the testing of production cell banks requires a much greater level of characterization than non-production banks. MCBs are the starting material for the entire production process. The regulatory expectation is that these are fully characterized for the microbial organisms already discussed, as well as for viral agents, including adventitious agents. This testing is performed one time only, unless changes are made to the bank, in which case the testing should be repeated with the new cells. As WCBs are only a small number of passages beyond the MCB, a reduced package of testing is acceptable. This reduced package typically includes sterility, mycoplasma, and minimal adventitious viral testing. Cells at the limit of 

 cell age (CAL) or EOP cells are considered to be the worst-case scenario for amplification of any contaminants present. These cells also require one-time full characterization testing. Refer to 

 for a summary of the regulatory testing expectations for each type of cell bank.

Table I: Summary of the regulatory testing expectations for each type of cell bank. MCB=master cell bank, EOP=end-of-production cell bank, WCB=working cell bank.

	Like the microbial agents already discussed, viral agents may be introduced through starting materials, such as cells or raw materials, as well as from operators or contaminated facilities. Raw materials such as bovine serum and trypsin continue to be common sources of viral contaminants in the industry. It’s crucial to understand the entire history of the cell bank and perform a thorough risk assessment when designing the viral-testing plan. Has the cell line been exposed to serum or trypsin? If so, was it irradiated? While the animal-based raw materials may no longer be used in the production process, if these reagents were used in the past history of the cells, they are still a concern and something that must be addressed in the testing plan. Was the cell line exposed to other cell species or substances derived from other species? What non-animal additives have the cells been exposed to? What viruses is the cell line susceptible to? These are all important questions that should be considered when performing the risk assessment and developing the testing plan.

	Characterization testing for viral agents includes both general, broad-screening assays that are intended to detect unknown viruses as well as tests for specific viruses. The general, broad screening tests are implemented to detect an unknown and wide range of possible contaminants. These tests include 

 virus assays, and electron microscopy. There are also a number of assays available that detect contaminants associated with specific species of concern; the bovine and porcine screening tests, which are performed to identify contaminants from serum or trypsin, as well as human or rodent screening assays based on product-specific concerns. There are also a number of assays to detect the presence of retroviruses, which may be both adventitious and/or endogenous (viruses that are known to integrate into the host-cell genome).

 virus assays, multiple indicator cell lines are selected and used to detect viral contaminants. The cells are inoculated with a cell-lysate sample and observed for indications of viral infection (cytopathic effects [CPE], hemadsorption of red blood cells [HAD], hemagglutination of red blood cells [HA]) over the course of several weeks. When selecting indicator cells to use in the testing, it’s important to select them based on their theoretical susceptibility to the viruses of concern. A human diploid cell line (MRC-5) and a primate cell line (Vero) are always used to detect viruses that are infectious for human cells. A third indicator cell line of the same or similar cell type as the cell bank substrate being tested is selected to detect viruses that are infectious to those cells. Additional cell lines may be added to address particular pathogens of concern. For example, A9 or 324K cells may be added to the testing panel to detect murine minute virus (MMV), a common industry contaminant. As with sterility and mycoplasma testing, it’s recommended that matrix interference testing be performed prior to routine testing to assess any sample inhibition or toxicity issues.

virus assays are also broad screening assays performed to detect unknown viruses. The rationale is that some viruses that don’t cause CPE or other noticeable effects in a cell-culture system may be detected in an animal system.

 testing involves inoculation of cell lysate into suckling and adult mice, guinea pigs, and embryonated chicken eggs (allantoic fluid and yolk sac). The animals are then observed for clinical signs every working day of the test.

	Testing for species-specific panels of viruses is also an important addition to the virus testing plan, and their use is based on the risk assessment. The most common species-specific testing includes testing for bovine and/or porcine agents, most often due to current or past exposure to serum and/or trypsin, respectively. The testing involves inoculation of indicator cells with cell lysate sample and observation of the cells over several weeks for CPE and HAD. The cells are then stained for the presence of a number of specific bovine and/or porcine agents as outlined by the 

 and European Medicines Agency testing requirements.

	For biopharmaceutical products prepared in rodent cells, the presence of rodent viruses is a significant concern, as many rodent viruses are able to replicate human cells. In these instances, antibody production tests can be performed, which are sensitive methods for the detection of adventitious rodent viruses. Viral antibody-free animals are injected with the test article and serologic analyses are performed at the end of the incubation period to determine whether antibodies were produced against specific viruses. Tests may be performed in mice, rats, or hamsters.

	It may also be important to consider the addition of virus-specific real-time polymerase chain reaction (qPCR) assays to the testing panel to detect specific pathogens that aren’t detected in the assays described previously. PCR panels specific for detection of simian, human, canine, avian, or insect viruses are also available.

	The last area of concern for cell characterization risk assessment is retrovirus testing. While these agents can be adventitious, many are endogenous to rodent cell lines. Because retroviruses are associated with serious immunosuppressive and degenerative conditions, they are a major safety concern. Therefore, it’s important to demonstrate the absence of infectious retrovirus. Retroviruses have the ability to remain latent within cells and can infect and replicate in the absence of obvious cytopathology, HA, or HAD. Specific co-cultivation procedures are necessary to detect infectious retroviruses. In all of these assays, susceptible target cells are co-cultivated with the cell line of interest. The target cells are subpassaged to allow for amplification of any retrovirus present, followed by detection of infectious virus in a secondary plaque assay for infectious virus or a PCR test to detect viral reverse transcriptase enzyme.

	Testing for adventitious agents is crucial for ensuring the safety and purity of a cell bank and resulting biopharmaceutical product. It’s important to perform a thorough and accurate risk assessment to prepare a testing plan and to allow for sufficient time to complete the testing, as many of the tests take weeks/months to complete. 

 shows a summary of standard industry timelines for the various tests. When considering the testing results, it’s also important to consider the implications of the assay results as well. For example, cell-based and animal assays detect infectious virus. In addition, many of these include amplification processes to increase the sensitivity of the assay. Molecular assays, however, detect gene sequences rather than infectious virus and may lead to a “false-positive” result by detecting a virus fragment that may not lead to infection. While it’s important to know the results of both cell-based assays and molecular assays, it’s also important to keep the results of the tests in context.

Table II: Summary of standard industry timelines for various cell-banking tests. qPCR=real-time polymerase chain reaction, StandardTAT=standard turnaround time.


	Genetic stability testing is a key component of production cell bank characterization and is a regulatory requirement. Typical mammalian production cell lines are created by stable transfection of the expression vector into the host cell line. During subsequent cell culture, genomic events such as deletion, rearrangement, and point mutation may occur and result in an altered cell phenotype and/or gene expression profile. The instability of the cell line is of great concern because it may negatively affect product integrity, thus posing a risk to the patients. Even when product integrity is not affected, possible reduction of productivity and elevated risk of future events are still of great concern from an operational perspective.

	Genetic stability testing typically includes an array of assays performed on the manufacturer’s MCB, EOP, and often WCB. Typical assays include, but are not limited to, those intended to confirm the integrity of the product transcript (mRNA/cDNA sequencing and Northern analysis), the genome structure at integration site (restriction digestion map via Southern analysis), and the ratio of insert gene copy number relative to host genome (via qPCR or Southern analysis). More recently, whole-genome sequencing using next-generation sequencing technology has been discussed within the industry as a potential method to confirm genetic stability and to further enhance cell line characterization.

	In addition to genetic stability testing, another important cell bank characterization test is the identity test. Until recently, identity testing was most often performed using isoenzyme analysis, which offered general speciation based on the migration pattern of a selected set of enzymes in the cell lysate when run on an agarose gel. The methodology had several known limitations, such as low sensitivity and difficulty with data interpretation. In addition to the technical limitations, a current shortage of the single-sourced test kit has highlighted the urgent need for alternative identity test methods. Among the possible alternatives, the cytochrome oxidase I barcoding method has become the front runner, due to high interspecies specificity and the availability of an extensive sequence database. For further authentication of human cell lines, short tandem repeat analysis is the preferred method and has been promoted by the American Type Culture Collection.

	Both genetic stability testing and identity testing should be performed on each MCB and on one representative lot of EOP cells. Genetic stability testing is optional for the WCB but may be performed at the manufacturer’s discretion. Genetic stability testing results from the EOPC and WCB are compared with those of MCB to allow the detection of any changes that may be indicative of instability. When changes in the manufacturing process are introduced, genetic stability testing may need to be performed on an additional lot of EOP cells.

 When referring to this article, please cite it as L. Mogilyanskiy, H. Byer, and W. Wang , "Mastering Cell Bank Production," 

Upstream Processing

A thorough cell-bank testing plan is necessary to certify the safety and purity of a resulting biopharmaceutical product.

Mastering Cell Bank Production

The authors explore the use of statistical experimental design and multivariate analysis to develop a drug substance formulation matrix.

	Protein aggregation is a major issue that can occur at any stage of biologics production and storage and often results in delays in the commercialization of protein drug candidates (1–3). To date, the mechanism of protein aggregation is still not well understood. The control and analysis of protein aggregation is a major challenge for many biopharmaceutical companies. While it is possible to reduce the risk of protein aggregation by optimizing buffer matrix, protein concentration, operating/storage conditions (i.e., temperature, agitation, contact material, etc.), and adding stabilizers, the measures to effectively preventing protein aggregates differ experimentally on a case-by-case basis. The presence of aggregates in drug products is undesirable, as it can stimulate immunogenic reactions and cause drug rejection upon administration to patients (4). In recent years, protein aggregation in drugs and drug candidates has gained increased scrutiny from worldwide health authorities (5–8). It is important to develop strategies to minimize protein aggregation early in process development and have proper analytical tools for monitoring, control, and characterization.

	Frozen storage is an important preservation method for soluble proteins; however, freezing (and thawing) of a protein solution introduces complex changes to the buffer environment that may lead to protein aggregation. Such changes include formation of ice-water interfaces, adsorption to container surfaces, cryoconcentration of protein and solutes, pH changes, and phase separation (1, 2, 9). Cryoprotectants have been used to reduce freeze/thaw (F/T)-induced protein aggregation. Many cryoprotectants are rich in hydroxyl groups that replace water molecules to form hydrogen bonds with protein molecules needed to maintain the high-order structure during freezing. Cryoprotectants also function by delaying ice crystallization and eutectic transition of a protein solution (10).

	In this study, protein aggregation was observed during F/T of a protein drug substance. To minimize the formation of F/T-induced protein aggregates, the effects of buffer, protein concentration, storage temperature, and cryoprotectants on protein aggregation, potency, and activity were investigated using dynamic light scattering (DLS), size-exclusion chromatography (SEC), potency, and activity assays, respectively. The operating ranges were optimized using statistical experimental design to establish the parameters and control limits for the drug substance formulation matrix. Finally, the drug substance formulated in the proposed matrix was subjected to multiple F/T cycles to confirm the acceptability of the optimized conditions.


	Protein aggregation was monitored using DLS and spectrophotometric methods. A number of conditions were screened, including buffering species, pH, salt concentration, protein concentration, cryoprotectants, cryoprotectant concentration, and storage temperature. Based on the screening results, the drug substance was optimized using a custom design-of-experiments (DOE) study, which refined the parameter ranges based on the responses of critical quality attributes of the drug substance (i.e., size-exclusion chromatography [SEC]-based purity, potency, and activity). The DOE results were used to create multivariate regression models for each of the responses. A prediction profile was constructed based on the multivariate regression models, which, along with the historical manufacturing data, were used to perform Monte Carlo simulations to identify process capability and potential number of failures (11). All the statistical experimental design and data analysis were performed using JMP software version 9.0 (SAS Institute, Cary, NC). Based on the DOE and Monte Carlo simulation results, targets and control limits for protein concentration and drug substance matrix components were determined. The operating ranges were confirmed by subjecting the drug substance to multiple F/T cycles.


	Polyethylene glycols (PEGs) of different molecular mass were used in this study. PEG-3350 was purchased from Dow, while PEG-4000 and PEG-6000 were from Fluka. Polyoxyethylene glycol dodecyl ether (BRIJ-35), PEG-300, and Tergitol NP-40 (nonylphenol ethoxylate) were purchased from Sigma-Aldrich. Poloxamer 188 (P-188) was from BASF. Human serum albumin was purchased from Talecris. Tween-20 and Tween-80 were from Croda. Sucrose and ammonium sulfate were from EMD Millipore, and other chemicals used in buffers were purchased from Mallinckrodt or J.T. Baker.

	DLS measurement was performed using a Zen 3600 Zetasizer (Malvern Instruments), and protein concentration was measured with a DU720 UV/vis spectrophotometer (Beckman Coulter). Potency was determined using a cell-based assay, while the proteolytic activity was measured based on a enzyme-based assay. Both potency and activity were reported as relative to a reference standard. Purity was assessed by analytical SEC, which detects aggregates and low-molecular-weight proteins.


	The presence of protein aggregates was measured before and after the F/T cycles were monitored by DLS, which determines the size distribution profile of small particles in Brownian motion in a solution. The drug substance solutions, in either citrate or phosphate buffer, were used to screen solution conditions that prevented F/T-induced aggregation. To determine the effect of protein concentration, buffer species, pH, salt, cryoprotectant, and storage condition on the prevention of F/T-induced aggregation, a series of screening experiments were completed; see 

 for a summary. Based on the initial screening studies, it was determined that of the factors investigated, only addition of cyroprotectants prevented F/T-induced aggregation.

 shows typical results from the screening studies. Under refrigerated conditions (2–8 °C), the drug substance retained the monomer. However, following a single F/T cycle, protein aggregates were detected (see 

, using DLS to detect aggregation, varying buffer species (acetate, citrate, or phosphate), protein concentration, pH, or salt level did not prevent F/T-induced protein aggregation. In contrast, many cryoprotectants were found to be effective in preventing F/T-induced aggregation, including Tween 20, Tween 80, P-188, NP-40, BRIJ-35, and all of the PEGs (see 

 for an example using PEG). As little as 0.1% PEG in either citrate or phosphate buffer inhibited the formation of aggregates.

	PEG is widely used in the pharmaceutical industry, readily available, easily dissolved, and does not interfere with UV absorbance. In addition, as a compound on FDA’s compounds generally regarded as safe (GRAS) list, PEG has low immunogenicity and itself is widely used as a drug (12). For this study, a medium-sized PEG was selected as the cryoprotectant, and only PEG results are presented hereafter.


	To assess the functional impact of the buffer or presence of PEG on drug substance potency--a critical quality attribute--the drug substance was prepared in replicates, in either citrate or phosphate buffer, supplemented with 1% of either PEG or blank buffer, and then tested for potency (

). The potency data were analyzed by one-way analysis of variance (ANOVA) and a Tukey-Kramer HSD [honestly significant difference] test (

). The results indicated that the buffer and PEG addition did not make a statistically significant difference on drug substance potency, at the significance level (α) of 0.05.


	To establish the ranges of drug substance matrix components, a multilevel, custom-designed DOE study with five factors and three responses was performed. The DOE evaluated the effects of ammonium sulfate concentration (0, 100, 140, 180, and 300 mM), pH (5.5, 6.0, 6.3, 6.5, 6.7, and 7.0), PEG concentration (0.1%, 0.5%, 1.0%, and 1.5%), protein concentration (multiple concentrations from 0.04 to 0.49 mg/mL, based on UV measurement results), and hold time (0, 2.5, and 5 days) on SEC purity, potency, and activity. A total of 32 runs were completed, and the results were analyzed using JMP Version 9.0. One run was identified as an outlier by Mahalanobis distance outlier analysis and was excluded from the calculations.

	Multivariate analysis was performed to identify the high level correlations among the factors. Linear regression models for each response were constructed using terms of individual factors, two-level interactions, and quadratics. The models were further refined by stepwise regression, taking away terms with p-values greater than 0.05. The refined regression models are illustrated in 

	The results indicated significant regression models for SEC purity (Panel A, adjusted R

: .81, p-value: <0.0001), potency (Panel B, adjusted R

: .67, p-value: 0.0024), and activity (Panel C, adjusted R

: .71, p-value: 0.0011), at α=0.05. The effect sizes and margins of parameter terms on responses are summarized in 

. Protein concentration, PEG concentration, and their associated interaction and quadratics have the biggest effect sizes among the different model terms.

	Scenario 1 used the proposed parameter targets, and the parameter standard deviation (SD) was calculated by dividing the proposed parameter range by six (i.e., the proposed parameter range was treated as the six sigma [6 x SD, or 6σ] range). Scenario 2 used the actual parameter targets and SDs from historical manufacturing batches. Scenario 3 used the actual parameter targets and SDs from historical manufacturing batches, with random noise adjusted so that the variation of predicted results matches that of manufacturing batch results. For simplicity, hold time was fixed at 0 day for the simulation as no hold is proposed at this step. The distribution of 10,000 simulated runs are compared with the distribution of the historical manufacturing batches in terms of mean, SD, failure rate (in % and ppm), and process capability (C

 illustrates an example of simulation profiles under scenario 2 and the simulation results are summarized in 

, under the different simulation scenarios, are all greater than or equal to 1.1.

 illustrates the predicted response surface under the different protein concentrations and PEG concentrations. The rectangles filled with blue color represent the proposed operating ranges. Based on the results, parameter targets and ranges were established for the drug substance formulation matrix.


	To confirm the selected conditions, drug substance solutions in the proposed formulation matrix were frozen at -70 °C or -150 °C. Samples were subjected to multiple F/T cycles prior to analysis by DLS. As shown in 

, repeated F/T cycles did not result in the development of aggregates, as detected by DLS.


	The objective of this study was to develop an appropriate drug substance matrix to prevent the formation of F/T-induced protein aggregates. In this study, freeze/thaw-induced protein aggregation was observed during process development. To minimize protein aggregation, PEG-3350 was added to the drug substance matrix, and the matrix formulation was optimized by a custom DOE study. Based on the DOE results, multivariate regression models were established, Monte Carlo simulations were performed under different scenarios, and drug substance matrix conditions were proposed. The proposed conditions were confirmed by experiments with multiple F/T cycles. Several batches of drug substance have been produced under the proposed conditions and have demonstrated good stability after storage.

 289, pp. 1–30 (2005).
	2. H-C Mahler et al., J

 98 (9), pp. 2909–2934 (2009).
	3. M.E.M. Cromwell, E. Hilario, and F. Jacobson, 

 8 (3), pp. E572–579 (2006).
	4. A.S. Rosenberg, AAPS J. 8 (3), pp. E501–507 (2006).
	5. FDA, 

Guidance for Industry, Immunogenicity Assessment for Therapeutic Protein Products

 (Rockville, MD, Aug. 2014).
	6. R.V. Cordoba-Rodriguez, 

 21 (11), (2008), accessed May 9, 2012.
	7. T.M. Scherer et al., 

. 14 (2), pp. 236–243 (2012).
	8. USP, USP General Information <1787>, “Measurement of Subvisible Particulate Matter in Therapeutic Protein Injections,” 

39 (2), March–April (2013).
	9. W. Wang, S. Nema, and D. Teagarden, 

 390 (2), pp. 89–99 (2010).
	10. Y. Mi, G. Wood, and L. Thoma, 

 6 (3), pp. 1–10 (2004).
	11. SAS Institute Inc., “

,” Cary, NC, accessed May 9, 2012.
	12. J. Chen et al., 


	Article submitted: Apr. 10, 2015.
	Article accepted: May 13, 2015. 


	Hui Xiang is principal scientist; Derek Chan is principal scientist; both at Allergan. Ronald Bates was director, at Allergan, Inc., and is currently director at Bristol-Myers Squibb.

 When referring to this article, please cite it as H. Xiang, D. Chan, and R. Bates, "Minimization of Freeze/Thaw-Induced Protein Aggregation and Optimization of a Drug Substance Formulation Matrix," 

Peer-Reviewed Research

Minimization of Freeze/Thaw-Induced Protein Aggregation and Optimization of a Drug Substance Formulation Matrix

	An important area of focus for the biopharma industry is the elucidation and development of robust, efficient, and economic purification strategies for new monoclonal antibodies (mAbs) coming through the pipeline. Since the first therapeutic antibody came onto the market in 1986, up to the end of 2014, 47 products have been approved in the United States or Europe for a number of diseases including various types of cancer, autoimmune, autoinflammatory diseases, and rare diseases (1, 2). A study by BioProcess Technology Consultants estimated that approximately 70 mAb products could be on the market by 2020, which could be associated with up to £125 billion in sales (1).

	Purifying these biological products once produced by cells requires a reliable and optimized chromatography process. An advantage in the area of mAbs is the possibility of using a purification platform approach. The typical platform process is generally comprised of an initial capture step with Protein A-chromatography, a first polishing step with cation-exchange media, and a final polishing step using anion-exchange or multimodal media. Even this platform process requires some fine-tuning and adjustment for new mAb targets, especially at the first polishing step with cation exchange chromatography in bind-and-elute mode. This step often requires more optimization than initial capture and/or final polishing and aims to achieve high-dynamic binding capacity and maximum aggregate and host-cell protein (HCP) clearance. In addition to assessing factors and conditions for the optimization of the purification steps, the quality-by-design (QbD) initiative introduced by FDA requires a deeper understanding of critical process parameters to be obtained during process development. This deeper level of understanding could be directly addressed by increasing the experimental space using high-throughput process development (HTPD). 

Leveraging the power of HTPD and DoE for chromatographic optimization


	The goal in HTPD is to evaluate numerous factors or conditions in parallel using micro-scale formats. It reduces experimental time frames from weeks to hours, requires low sample consumption, and increases process understanding by exploring a larger experimental window. With HTPD, the typical workflow follows a series of steps, including planning an experimental setup using a systematic approach, carrying out experiments by testing various experimental conditions in a parallel manner, analyzing data using high-throughput analysis, and evaluating data and analyzing the results.

	Design of experiments (DoE) provides a statistics-based methodology to identify and define factors affecting the overall process or end product. By studying multiple factors simultaneously, DoE allows a maximum amount of information from the minimum number of experiments. Objectives such as screening, optimization, and robustness testing are set before the studies begin. Data generated by DoE can be used to create models that can apply statistics to predict future responses of a particular system to conditions that so far have not been tested. An example illustrating the use of HTPD and DoE to optimize the first polishing step for a mAb of interest is discussed herein, and the roles of both HTPD 96-well filter plates and mini-columns are highlighted (3).

Case study: Accelerating mAb purification via HTPD


	The mAb used in the following case study was produced in Chinese hamster ovary (CHO) cells, and the initial mAb capture step was carried out with an alkali-stabilized, Protein A-derived affinity medium using a standard protocol. An HTPD approach was then taken for the subsequent purification using a high-capacity cation exchanger. First, static binding capacity (SBC) was determined, using 96-well filter plates to allow high-throughput screening of a large number of experimental conditions. 96-well filter plates prefilled with chromatography medium (GE Healthcare’s Life Sciences business) were run in batch mode. In HTPD, simple absorbance measures such as A280 measurements can be used, and additional analyses carried out for the most critical conditions. In this mAb case study, SBC increased by increasing pH from 4.5 to 6.0 and the salt concentration from 0 to 50 mM NaCl (see 

). Further investigation of binding capacity and selectivity was limited to the known pH limits for the stability of the mAb being tested.

Figure 1: Results of static binding capacity (SBC) screening are displayed in a contour plot, as determined using a high-throughput process development (HTPD) 96-well plate prefilled with a strong cation-exchange chromatography medium. 

	Dynamic binding capacity (DBC) was assessed using high-throughput modular mini-columns (GE Healthcare). By using mini-columns operated in parallel, a larger number of different experimental conditions, such as binding, and/or wash and elution conditions, can be explored in a shorter time frame compared with when using small-scale columns, which are operated in a sequential mode. In this study, a robotic liquid handling system was used to run eight columns, packed with the cationic exchange medium, in parallel. Using a DoE approach, different pH and NaCl concentrations were studied to determine the dynamic binding capacities at selected conditions. A mAb eluate from the Protein A capture step conditioned to the different pH and salt concentrations was loaded onto the columns. Protein concentration in flowthrough fractions were analyzed by UV spectroscopy. DBC was found to increase with pH and NaCl concentration within the explored space, which correlated well with the SBC experiment (see 


	Table I: Effects of different binding and elution conditions on monoclonal antibody (mAb) aggregate and host-cell protein (HCP) content at 90% monomer yield. The center point of the factors is halfway between the lowest and highest levels (pH=5.5, NaCl=25 mM). Selectivity between mAb monomer and aggregates was also studied using DoE, analyzing factors such as pH and NaCl concentration. For this purpose, small-scale columns were employed. Aggregate concentration and host-cell protein (HCP) levels in the eluate at a 90% monomer yield were observed. The loading pH was found to have a significant effect on aggregate concentration, where lower pH was optimal for a low level of aggregates. A 2–4 fold reduction in HCP was observed following the cation exchange polishing step (see 

	The result of process optimization was a DBC of >100 mg mAb/mL medium at a loading condition of pH 5.0 and 50 mM NaCl. A Tricorn 5/100 column (GE Healthcare) was used to verify the DBC at 10% breakthrough (Q

), which was 109 mg mAb/mL, comparable to the mini-columns used (108 mg mAb/mL). With a load of 50 mg mAb/mL, less than 1% aggregate content was found and monomer yield was above 90%. Even at a higher sample load of 76 mg mAb/mL, 0.9% aggregate at a monomer yield of 93% was achieved, with an HCP concentration of 170 ppm and less than 1 ppm of leached Protein A from the capture step (

 Figure 3: Selectivity between monoclonal antibody (mAb) monomers and aggregates at a higher sample load following the optimized first polishing step in a lab-scale column, showing the percentage of fragments and aggregates in the resulting fractions. A280=absorbance at 280 nm wavelength as measured with UV-visible spectroscopy; mAU=1/1000th of an absorbance unit as measured with UV-visible spectroscopy. 

Considerations when using mini-columns on a robotic liquid-handling station


	The slowest flow rate when using a 1-mL syringe on the TECAN Freedom Evo 150 robot, for example, is 1 µL/s. With PreDictor RoboColumn units 200 µL (GE Healthcare), this flow rate will result in a residence time (RT) of 3.33 minutes. Using the larger column volumes (i.e., 600 µL) will increase the RT to 10.0 min with the slowest flow rate. Thus, depending on what residence time that is needed, the relevant column volume should be selected.

	To avoid system liquid dilution of the protein sample during sample injection to PreDictor RoboColumn units, an excess volume of 400 µL protein sample needs to be aspirated. This means that with the 1000-µL syringe, 1000 µL of protein sample can be aspirated, but only 600 µL will be dispensed into the columns. The issues with dilution have not been observed for buffers and salt containing aqueous solutions.


	The dynamic binding capacity is dependent upon several experimental parameters (e.g., the charge and size of the target molecule, pore size, and the charge of the chromatography media, as well as the ionic strength of the loading buffer). The highest DBC on an ion-exchange chromatography media is typically expected at low conductivity and at a pH far away from the isoelectric point (pI), where the protein charge is high. For some proteins, increasing conductivity and decreasing protein charge can improve capacity (4, 5). Careful investigation of the effect of pH and salt concentration on dynamic binding capacity should be undertaken in order to find the highest binding capacity.


	The productivity, expressed as amount of product per unit of time and unit of chromatography media, is determined by the breakthrough capacity of the chromatography media, the recovery of active product, and the cycle time.

	The cycle time can be reduced by selecting modern chromatography media that can be operated under high volumetric flow rates. Even though the binding phase may be constrained to a certain residence time due to mass transfer limitations and a decrease in capacity at higher flow velocities, equilibration, wash, and elution can be carried out at maximum allowed operating velocity.

	Optimization of each phase in a chromatographic cycle to decrease the cycle time will have a positive impact on productivity. By transferring a continuous gradient elution to a step gradient elution, for example, there is a significant decrease in elution volume, which shortens cycle time and increases productivity.


	The use of a HTPD-based workflow in conjunction with DoE has been described as a method to optimize conditions for the first polishing step of mAb purification. This workflow is compatible with robotic liquid handling stations for 96-well filter plates and mini-columns and steps can be taken to reduce the cycle time while increasing productivity, including improved recovery of active products.

	Plates and mini-columns, however, have different advantages and limitations for various applications. When used in combination, they provide a powerful platform for quickly and efficiently determining which conditions work best for a particular mAb of interest, with excellent correlation and reproducibility at larger scale. Filter plates are useful tools to screen for a broad range of conditions, whereas mini-columns can be used to investigate dynamic parameters. Lab-scale columns can then be used to follow-up from the high-throughput systems to determine selectivity and verify binding conditions.


	1. D.M. Ecker, S.D. Jones, and H.L. Levine, 

 7 (1), pp. 9-14 (January/February 2015).
	2. A. Beck et al., 

 10, pp. 345-352 (May 2010).
	3. GE Healthcare, “Optimization of dynamic binding capacity and aggregate clearance in a monoclonal antibody polishing step,” Application note 29145068 AA (2015).
	4. C. Harinarayan et al., 

 95 (5), pp. 775-787 (2006).
	5. GE Healthcare, “Screening and optimization of the loading conditions on Capto S.,” Application note 28-4078-16 AA (2006).

	Anna Grönberg is staff research engineer, R&D, at GE Healthcare.

	ALL FIGURES ARE COURTESY OF THE AUTHOR. 

When referring to this article, please cite it as A. Grönberg, "Optimizing Cation-Exchange Chromatography with High-Throughput Process Development for mAb Purification," 

Troubleshooting

Optimization of each phase in a chromatographic cycle has a positive impact on productivity.

Optimizing Cation-Exchange Chromatography with High-Throughput Process Development for mAb Purification

	As concerns mount around the world regarding adulterated and counterfeit drugs and medical products, FDA is joining manufacturers and other regulatory bodies to take more aggressive action against “fake pharma” operators. There were more than 2000 incidents of pharmaceutical crime in 2014, according to the Pharmaceutical Security Institute (PSI); these involve diversion, theft, and counterfeiting in more than 100 countries, primarily in Asia.

FDA and industry support global framework and collaborations to secure the supply chain.

BioPharm International-08-01-2015

Drug Delivery Systems for Biopharmaceuticals

Selecting a Comprehensive Bioburden Reduction Plan

Mastering Cell Bank Production

Minimization of Freeze/Thaw-Induced Protein Aggregation and Optimization of a Drug Substance Formulation Matrix

Optimizing Cation-Exchange Chromatography with High-Throughput Process Development for mAb Purification

Campaign Against Fake Drugs Gains Momentum

Outsourcing Becoming More Cost Competitive

Finding a Cure for Early Pipeline Failures

Cleaning of Dedicated Equipment: Why Validation is Needed

Fluid Handling in Biopharma Facilities

BioPharm International, August 2015 Issue (PDF)

Compliance with US and EU Internal Audit Requirements