Feliza Mirasol

	Albumin, the most prevalent protein found in blood plasma, is an emerging drug-delivery tool that has been found to be versatile and effective. Albumins can be used in drug formulation because they have been shown to stabilize and extend the shelf-life of the end product. Furthermore, albumins, both naturally occurring and engineered, can be used to extend the half-life of a drug.

Pharmaceutical Technology’s Biologics and Sterile Drug Manufacturing 2018

	When referring to this article, please cite it as F. Mirasol, "Albumin Attachment Can Make Better Biobetters," 

Pharmaceutical Technology Biologics and Sterile Drug Manufacturing 

Articles

Naturally occurring and engineered albumins are being explored as a tool to enhance the stability of drugs, including biologics, and extend shelf-life.

Albumin Attachment Can Make Better Biobetters

Stability testing is essential for maintaining the integrity and quality of biopharmaceuticals and for assessing an accurate shelf-life. It is an important aspect of quality control and is an important step in evaluating product safety and efficacy. It is also important for examining how critical quality attributes (CQAs) of a drug substance vary with time under different environmental factors.

	Through stability testing, pharmaceutical companies can ensure the most suitable packaging and/or container closure for the storage and distribution of biopharameutical products, according to Russell Crothers, supervisor, Sample Control Unit, Alcami.

	“With the right storage, appropriate shelf life determined, and distribution methods in place, the quality of active pharmaceutical ingredient (API) and drug products is safeguarded,” Crothers says.

	In addition, understanding potential degradation routes in relation to storage environment is an important factor in establishing the CQAs of a pharmaceutical or biopharmaceutical, according to Ashleigh Wake, director, Biological Services, Intertek Pharmaceutical Services. “Ultimately, this understanding ensures that the optimal quality control strategy is in place to monitor the continued efficacy and safety of any therapeutic,” Wake says.

	“Imagine the situation where, in the first instance, the degradation route of a molecule had not been assessed through forced or ‘stressed’ studies.  Not understanding this pathway could lead to failure in identifying pertinent degradation products [that] impact the safety and/or efficacy of the product and thus need to be controlled during product release,” says Wake.

	Wake goes on to note that if the potential formation of impurities is not evaluated prior to the release of the product, then methods will not be in place to monitor levels. This could lead to a rise or change in profile, which might not be detected until an “effect” in patient population is observed.

	“More so, without understanding the stability both of drug substance and drug product, the shelf life cannot be effectively established. This may not only risk the safety and efficacy of a product supplied to patients but can lead to unrealistic or even untenable pricing, especially for biologic molecules, which are expensive to produce, thus impeding their availability,” she adds.

	There are several important factors to consider when designing and conducting stability studies.

	“Safety, quality, and product efficacy work together in stability studies of APIs and finished drug products. Through stability testing, pharmaceutical companies have the ability to identify and trend shelf life and their effects on efficacy as samples are exposed to time, light, and temperature,” Adam Keisker, supervisor, Laboratory Support Services,  Alcami, states.

	Degradation factors that are fundamental to consider for the efficacy and shelf life of APIs/finished drug products include physical, chemical, and microbiological factors. Physical factors encompass changes to the physical nature of the drug, such as appearance, properties, hardness, brittleness, and particle size, that occur in tablets, capsules, and semisolids.

	From a chemical perspective, scientists look for separation of the chemical compound into elements or simpler compounds or a change in the drug’s chemical nature via hydrolysis, oxidation, isomerization, polymerization, or photodegradation, according to Keisker.

	Finally, microbiological contamination of a product, depending on the type of microbe and its level of toxicity, can also play a role in the design and functions of the studies, he adds.

	The strategy used for testing product stability is the most important consideration in the design of a pertinent study, according to Wake.

	“If, prior to a formal stability evaluation, work is not performed to understand all potential degradation routes of the molecule through stressed studies, methodologies cannot be effectively chosen for inclusion in the formal study to encompass assessment of degradation product formation and thus all such species monitored,” says Wake.

	“For any drug substance or product, an effective stability study cannot be designed on a ‘tick box’ approach for analytical assessments to be included for testing at timepoints,” she adds.

	Wake adds that all stability programs should include methods to confirm identity, assay, purity, and impurities. “However, the choice of the actual analysis and number/mode of methods utilized cannot be a ‘standard set’, but should be designed on a bespoke basis.”

	“Choice of storage condition or conditions should also be carefully determined prior to initiation. [International Council for Harmonization (ICH)] Q1 and Q5C (1,2) provide guidance on study design for pharmaceutical and biopharmaceutical products and should be the basis for all design; however, the eventual program requires considered input,” explains Wake.

	As an example, Wake points out that, if the intended storage condition is refrigerated, this condition should be considered as the long-term storage condition to be evaluated for a minimum of 12 months, typically longer. An accelerated condition would then, in most instances, relate to storage at 25 °C/60% relative humidity (RH) for six months and, importantly, the need for assessment at 40 °C/75% RH, would not typically be required.  

	“Logistically, the amount of material, drug substance, and/or drug product required to support what can be as long as a five-year program with multiple time points needs to be considered at onset. Not only should this include a realistic amount for the defined program, but should incorporate a minimum of an additional 25% to act as back-up material in case of re-test,” according to Wake.

	“In addition, ICH guidance requires that a minimum of three batches of material be included in formal stability studies, again this can put pressure on sample requirement and timely availability,” she adds.

	Having a strategic approach and considering a plan for a “worse-case” scenario is important to addressing the challenges of stability testing and to conducting a successful testing program. It is also important to approach each study with a bespoke, considered design for storage and testing, rather than a “tick box” approach, Wake says.

	“Perhaps the biggest challenge is to ensure that the testing program incorporates sufficient analytics to ensure all potential and known degradants are continually monitored.  When considering complex molecules, such as biologics, the complexity of degradation and the number of potential routes makes this assignment extremely difficult,” Wake explains.

	If a potential impurity is missed and later seen to appear, this may detrimentally affect the integrity of the stability study. In a worst-case scenario, the missed impurity may require re-testing, which would introduce significant delay to product registration, Wake notes.

	“Knowing all the ways a finished product or API could be affected by degradation is crucial in performing successful stability tests. For instance, stability studies are executed to simulate climatic effects. The studies are based on where the products are going to be sold. From those studies, scientists are able to better establish a shelf life of the medicine, determine the best way to store the medicine, and ultimately help ensure the safety of the consumer,” says Keisker.

	Analytical methods for effective stability testing vary from drug product to drug product. The design of stability studies must take into account the product form, container type, and packaging, Keisker notes.

	“For example, commercial drug product testing would include studying the degradation effects from the conditions on both the drug product and the container it is sold in,” Keisker says.

	“There are many variables but it ultimately depends on the client and their interests as well as requirements for the climate zone they intend to sell their product,” he adds.

	In terms of methodology, most small-molecule pharmaceutical stability study programs are typically based around the need for some analysis that is dependent on the presentation of the substance or product, such as, for example, water content only being necessary on lyophilized or solid material, Wake says.

	Not all analytics will be required at each timepoint, their inclusion would be driven by the likelihood of change, for example, sterility is typically assessed at six-month intervals, unless a specific indication warrants more frequent analysis.

	In comparison, for biologics, the nature of the stability testing to be included in any program differs, driven by the complexity of biologic molecule structure. In many ways, biologics require a more diverse analytical capability, according to Wake. Defining a typical stability testing protocol is consequently more difficult, but as a minimum would include assessment of the quality attributes.

	“Other criteria such as process related impurities, solvents, metals, etc. are typically not included in a stability program but determined on product release. Again there can be exceptions where there is a potential for the amount of such species to increase on storage,” says Wake.

	In the case of multi-dose product types, in-use stability testing can be used. The intent of an in-use stability study is to simulate the use of the product in practice, taking into consideration the filling volume of the container, any dilution/reconstitution before use, the hold-time before use, and various diluents that could be used for administration, Crothers says. 

	The purpose of conducting in-use stability testing is to establish a period of time during which a multi-dose product may be used while retaining quality within an acceptable specification, once the packaging is open or broached, Wake says, citing the European Medicines Agency’s definition of the term (3).

	“In-use stability testing can therefore be considered for multi-dose product types, as assessment of the continued efficacy and safety (as defined through critical quality attribute testing) of a pharmaceutical (or biopharmaceutical) drug product once in its final administration form,” according to Wake.

	Typically, an in-use assessment is performed on a minimum of two batches of material, with, ideally, one batch taken from a near end-of-shelf-life product. The protocol involves reconstituting the drug product to its administrative form and testing over a pre-defined storage period at the condition recommended. 

	“The analytics performed will be in line with those identified for long-term and accelerated storage studies with perhaps a greater influence on microbial criteria and potential impurities derived from pertinent environmental factors, for example, impurities formed through oxygen exposure which is unavoidable in a multi-dose format,” according to Wake.

	Establishing an effective in-use shelf-life is of significant benefit. Without such, a multi-dose product design becomes significantly less practical in terms of ensuring patient safety and product efficacy in this format. “This can lead to huge product wastage which, specifically in the case of biological materials, has huge consequence in terms of product cost and maintaining supply which can lead to the product being non-viable in terms of patient access,” says Wake. 

, Step 5 version (ICH, 2003).
	2. ICH, Q5C 

Quality of Biotechnological Products, Stability Testing of Biotechnological/Biologic Products

Note for Guidance on In-Use Stability Testing of Human Medicinal Products


	Vol. 42, No. 3
	March 2018
	Page: 52–56

	When referring to this article, please cite as F. Mirasol, “Stability Testing Determines Proper Drug Storage Parameters,” 

Stability testing on APIs/finished drug product helps define optimal drug packaging for shelf-life storage.

Stability Testing Determines Proper Drug Storage Parameters

	In addition, understanding potential degradation routes in relation to storage environment is an important factor in establishing the CQAs of a pharmaceutical or biopharmaceutical, according to Ashleigh Wake, director, Biological Services, Intertek Pharmaceutical Services.

	“Ultimately, this understanding ensures that the optimal quality control strategy is in place to monitor the continued efficacy and safety of any therapeutic,” Wake says.

	“Safety, quality, and product efficacy work together in stability studies of APIs and finished drug products. Through stability testing, pharmaceutical companies have the ability to identify and trend shelf life and their effects on efficacy as samples are exposed to time, light, and temperature,” Adam Keisker, supervisor, Laboratory Support Services, Alcami, states.

	As an example, Wake points out that, if the intended storage condition is refrigerated, this condition should be considered as the long-term storage condition to be evaluated for a minimum of 12 months, typically longer. An accelerated condition would then, in most instances, relate to storage at 25 °C/60% relative humidity (RH) for six months and, importantly, the need for assessment at 40 °C/75% RH, would not typically be required.

	“Logistically, the amount of material, drug substance, and/or drug product required to support what can be as long as a five-year program with multiple time points needs to be considered at onset.  Not only should this include a realistic amount for the defined program, but should incorporate a minimum of an additional 25% to act as back-up material in case of re-test,” Wake says.

	“Perhaps the biggest challenge is to ensure that the testing program incorporates sufficient analytics to ensure all potential and known degradants are continually monitored. When considering complex molecules, such as biologics, the complexity of degradation and the number of potential routes makes this assignment extremely difficult,” Wake explains.

	Analytical methods for effective stability testing vary from drug product to drug product. The design of stability studies must take into account the product form, container type, and packaging, Keisker notes. “For example, commercial drug product testing would include studying the degradation effects from the conditions on both the drug product and the container it is sold in,” he says.

	“There are many variables but it ultimately depends on the client and their interests as well as requirements for the climate zone they intend to sell their product,” Keisker adds.

	In terms of methodology, most small-molecule pharmaceutical stability study programs are typically based around the need for some analysis that is dependent on the presentation of the substance or product, such as, for example, water content only being necessary on lyophilized or solid material, Wake says. Not all analytics will be required at each timepoint, their inclusion would be driven by the likelihood of change, for example, sterility is typically assessed at six-month intervals, unless a specific indication warrants more frequent analysis.

	Typical methodologies for certain quality attributes are illustrated in 

	In comparison, for biologics, the nature of the stability testing to be included in any program differs, driven by the complexity of biologic molecule structure. In many ways, biologics require a more diverse analytical capability, according to Wake. Defining a typical stability testing protocol is consequently more difficult, but as a minimum would include assessment of the quality attributes shown in 

	“In-use stability testing can therefore be considered for multi-dose product types, as assessment of the continued efficacy and safety (as defined through critical quality attribute testing) of a pharmaceutical (or biopharmaceutical) drug product once in its final administration form,” Wake says.

Volume 31, Number 1
	January 2018
	Pages 42–45

	When referring to this article, please cite as F. Mirasol, “Stability Testing Ensures Proper Packaging for Drug Storage,” 

Stability testing on APIs/finished drug product is crucial to ensuring proper packaging for optimum shelf-life storage.

Stability Testing Informs Drug Packaging Specs for Proper Storage

Criteria for choosing a partner or service provider for analytical services in early phase drug development are based on scientific skills/knowledge, resource availability, and customer needs.

	An analytical service provider for early phase development studies should show that they have the necessary scientific skills and knowledge to support the program. Discussions between client and provider are needed to ensure that both parties are comfortable with what is required out of the relationship, according to John Wood, analytical account manager, Almac Group.

	Wood notes that once the preliminary steps are established, other considerations should include:

		Resource availability-can the service provider provide the services within the required timeframe? A project plan should be requested detailing all activities and delivery dates.

		Ensuring clarity about what documentation is to be provided by the service provider and whether this is sufficient for any regulatory submissions.

		Whether the service provider will provide regular updates and the format of these updates--regular telephone conferences are recommended as communication is key to a good relationship.

		Whether the service provider can provide all services in-house or would parts be subcontracted. If subcontracted, does the service provider have a subcontract lab in place to perform these tasks?

	“There is any number of criteria used to select a service provider depending on the scope of work being requested,” says Roger Hayes, PhD, senior vice-president, Drug Metabolism and Pharmacokinetics, MPI Research, an early stage drug development contract research organization (CRO).

	“For example, a sponsor may have extensive in-house capabilities but lack a vivarium with non-human primates. In that case, the sponsor will seek a service provider with the appropriate animal model. On the other hand, a virtual company may look for a service provider that has all of the services under one operational ‘roof’,” he says.

	Once the required service is defined, the selection criteria hierarchy is schedule, quality, and then price. For more commoditized studies, the ordering may be price, schedule, and then quality, according to Hayes. “There are certain ‘table stakes’ for a service provider to be considered, and those usually relate to regulatory compliance (USDA [US Department of Agriculture] and FDA), scientific knowledge/experience of the study directors, communication, and prior experience or reputation,” he adds.

	“It’s been our experience that there are two key criteria for choosing a service provider: the collaborative relationship the service provider can build and foster with the client, and the ability of the vendor to provide a wide-range of services, including analytical, bioanalytical, in vitro and in vivo services, all the way through to enabling toxicology studies,” says Philip J. Kuehl, PhD, director of Scientific Core Laboratories, Lovelace Biomedical, a not-for-profit CRO. 

	Other criteria that Kuehl points out is the responsiveness of the service provider, including having strong communicative skills to make clients feel valued, the ability to meet timelines, and the capability to review and share good or bad data quickly and efficiently. “The service provider should also be able to make a scientific contribution to the data, as it applies to the interpretation and decision-making. It is helpful if they have a breadth of experience, including small- and large-molecule capabilities in and around analytical and bioanalytical services,” he notes.

	Simple one-off service agreements or short contracts are the most common relationships in early phase analytical support. These finite relationships fit well where the work required is reasonably well-defined and milestones are expected to be met in the near future. “This will guarantee resource availability and the analytical team can work flexibly to provide support as priorities may change and different molecules may get fast tracked ahead of other projects,” Wood says.

	The most frequently sought-after relationships on the service provider side, however, are strategic partnerships. “No CRO would be able to sustain a business with only transactional fee-for-service contracts. The operational overhead involved in a competitive bid process will drive a desire for a partnership with preferred pricing to entice the relationship,” says Hayes.

	Hayes points out that even repeat transactions gain some familiarity with a client over time, and there is typically a period over which the relationship transitions from transactional to preferred and finally to strategic. “Somewhat uniquely, bioanalytical CROs tend to attract loyal sponsors that then require a string of poor performance before the activation barrier to seek out a new provider is broached. Preferred partnerships create additional opportunities for both the sponsor and the CRO.  The sponsor has access to niche CRO talent that may otherwise go untapped if a relationship remains transactional, and the CRO has an opportunity to participate on research projects that may not typically be outsourced,” he says.

	It takes time to switch over to a strategic partnership, and this is not often required given that the service being offered is commoditized, Hayes explains. “The strategic aspect could be as simple as the client providing a peek into long-term needs to ensure that adequate resources are made available. Milestone discounting or rebates based on spend become the reciprocal enticement for the client.”

	The success of an outsourced relationship depends on the existence of a true partnership in drug development. These successful relationships involve the use of a service provider’s scientific breadth of expertise and critical decision-making abilities, notes Kuehl. Also, interpretation of results and the skills to move a program along efficiently so that the ultimate goal is achieved together plays a critical role in successful partnership. “However, there are always times when short-term of specific one-time service agreements are suitable for a program,” he adds.

	Analytical services that are typically the most sought after by clients during the early drug development phase include bioanalytical services and analytical methods to determine drug substance and drug product stability.

	“For preclinical and early phase development, the most important criteria are to develop analytical methods that are suitable for release testing of the drug substance and drug product and can be used to provide early stability data,” says Wood. “Method validation is not a priority for early phase molecules, but the methods should be shown to be suitable during the development phase.”

	“Bioanalytical services by far and away are the most sought-after analytical service, second only to the in-life studies that generate the PK [pharmacokinetic] samples for bioanalysis,” Hayes says. “Analysis of the dosing material is sometimes required in bridging studies and always required for the GLP [good laboratory practice] drug safety studies.  Analysis of the dosing material is most often sourced to the same laboratory providing the vivarium service, as there are significant benefits to having the dosing and the dose analysis in the same facility,” he adds.

	Most researches seek three characteristics in analytical services: adaptability, responsiveness, and translatability, according to Kuehl.

	“Customers look for a group that has the bandwidth to do things quickly, to be responsive and adaptive to the data as they are generated. For example, it’s great if a lab is able to start on a project relatively quickly. Then, as the data are generated, if there are changes in the assay, detection limit, matrix, or sampling process, the service provider will adapt and make changes quickly, in order to support the overall program. That’s where a strategic partnership is such a key part of the success of an analytical program--adapting to the needs of the overall program, not just a small scope of work,” Kuehl says.

	In addition, customers seek translatability, which is the ability to translate methods, assays, findings, and other required information from early proof-of-concept studies through to good practices regulated validated studies, Kuehl adds.


	Supplement: Partnerships in Outsourcing 2018
	February 2018
	Pages: s6–s8

	When referring to this article, please cite it as F. Mirasol, “Outsourcing Analytical Services in Early Drug Development," 

Pharmaceutical Technology Partnerships in Outsourcing 2018 

Successful outsourcing relationships in early phase drug-development analytics are driven by partnership.

Outsourcing Analytical Services in Early Drug Development

Partnership dictates the success of outsourcing relationships in early phase drug-development analytics.

Early Drug-Development: What to Consider When Outsourcing Analytical Services

The use of plastic bioreactor bags in single-use systems pose their own set of unique challenges for determining the presence and mitigating the risks of extractables and leachables (E&L) in the biomanufacturing process. What are these challenges and how can a manufacturer troubleshoot the problems that can affect product quality and efficacy?

	In an interview with Derek Wood, associate director, Extractable/Leachable and GC/MS Services, PPD Laboratories; James McLean, manager, Analytical Science, Catalent Pharma Solutions; Andreas Nixdorf, business development manager, SGS Life Sciences; and John Iannone, director, Extractables/Leachables & Impurities, AMRI, the vulnerability of these single-use bioreactor bags to E&L are discussed.

 Are single-use bioreactors, which are typically comprised of plastic, vulnerable to contamination by E&L material, and how vulnerable are they?

 Extractable/leachables evaluation for all types of components used in biopharmaceutical single-use system manufacturing components, including single-use bioreactors, is an important consideration and is a hot topic in the industry. 

	The advantages of the disposable manufacturing process for biologic production are well understood, but, unfortunately, many of the elastomeric and polymeric components used in such processes, such as plastic films used in bioreactors, are more likely to contribute impurities in the form of leachables compared to more traditional, fixed manufacturing equipment components. Leachables from single-use component materials may propagate to the final drug product and potentially present a direct health risk to the patient, but, even more likely for biologics manufacturing especially, a key concern is the potential for leachables to alter the quality and efficacy of the product itself.

	A single-use bioreactor utilizes a disposable bag in place of a fixed culture vessel. Such bags typically are constructed of multi-layer, polymer films, incorporating the use of a middle barrier layer (for example, ethylene vinyl alcohol) sandwiched between an inner film, such as polyethylene, that has direct contact with the cell-culture media, and an outer layer film designed to impart structure/strength and durability. The outer layer material may be, for example, a modified form of polyethylene, nylon, or polyester, and the three layers are bonded together in the material manufacturing process.

	The film material types for the different layers will vary based on the supplier. For example, in some cases, the inner layer may be polypropylene with polyvinyl alcohol  as the middle layer.

 The single-use bioreactor system (SUS) is more susceptible to extractables due to the nature of the materials of construction. These SUSs are designed to be highly flexible. In order to achieve the desired flexibility, the materials of construction tend to be polymers with lower levels of crystalline structure, such as low-density polyethylene (LDPE), which would facilitate higher diffusion of chemical species, such as plasticizers, antioxidants, thermal stabilizers, slip compounds, and monomers. This is further exacerbated by the intended use of the SUS.

	Historically, manufacturing and in-process equipment involved transient contact times with the API and/or drug product media measured in minutes to hours. For SUSs, their contact time may be days, or, in the case of intermediate bulk storage, years. These extended exposure conditions serve to make the extractable potential a higher risk.

 Plastic bags are often made from different film layers that can be made with different resins, each of which has a unique combination of properties that makes it ideal for certain applications. For example, low-density polyethylene film acts as a gas barrier, which is necessary for drug packaging because the drugs would quickly spoil if exposed to oxygen. Polyvinyl chloride film, on the other hand, is gas permeable, but an excellent barrier against moisture.

	Polyester films can be used as mono films for pharmaceuticals requiring low moisture barrier, or they can be integrated into barrier structures. Plastic films also can be clear or colored, printed or plain, single- or multi-layered and combined with other materials, such as aluminum and paper. For bag construction, multi-film constructions are mostly used. Thus, depending on the bag construction, leachables can be originated from different polymer sources.

 [Single-use] reactors are certainly vulnerable to imparting E&Ls into the contents of the bioreactor cell-culture medium. Depending on the additives that are needed for the bioreactor bag, there are a number of different compounds that can leach out and be present within the cell-culture medium. An example, which was published in a scientific journal (1), is a very well documented finding where Chinese hamster ovary (CHO) cells used in bioreactor bags, under a specific instance, were dying off.

	It was later discovered that, in the post-gamma-irradiation of the bag, a leachate derived from one of the antioxidant’s breakdown products was producing a compound that was responsible for killing off the CHO cells, even when the compound was present at very low levels. While it wasn’t of toxicological consequence to the patient, it still had a significant impact on bioproduction at that early stage and was affecting the efficacy of the process.  

	Another point to consider is that single-use-bioreactors are typically very large bags that expose an abundant surface area of the polymer to whatever it is that is in the bag. As the surface area of the material increases, so does the potential for leaching out various additives, breakdown products of the additives, breakdown products of the polymer itself, and different residues from the process.

	The high surface area certainly increases the vulnerability for this leaching to occur, although larger bags typically will result in a lower bag material surface area to content solution volume ratio. This ratio suggests the concentration of a leachate would be lower, regardless of the increased probability.

	The type of product that is contained within the bioreactors also can have a direct effect on the leachability of some of those compounds. Extraction propensity and solubility play a significant role in what will come out. Furthermore, time and temperature can drive leachates. When talking about bioreactors and cell culture, it is desireable to optimize the conditions for those cells to behave in the way they are intended to in order to produce the desired product efficiently. Typically, they operate with slightly elevated temperatures--about 37°C--to optimize cell proliferation, and with that slight elevation in temperature, they increase the potential for extractability. All of these different factors or parameters play a role in how vulnerable these systems are.

	One additional point to consider is that bioreactor bags tend to be more amorphous in nature in terms of the polymer. The more amorphous the polymer is, the more amenable it is to allowing leachates to diffuse from the polymer into its surrounding environment.

 At which stage(s) of the biomanufacturing process is testing done to determine the presence of E&L, and what can be done about it in real-time if detected?

 An effective risk management and risk mitigation is essential to the effective control of extractables associated with any biomanufacturing process. This should be a product control lifecycle strategy with equal focus on the three critical tiers of materials, container closure, and processing systems and drug products.

	For raw materials (both product-related and manufacturing), it is vital to ensure processes exist to monitor precursor material controls, material controls, service level agreements with vendors, periodic evaluation of control monitoring methodology, and a robust requalification program. For drug products, major considerations should be the drug product method development and validation, appropriate stability testing, toxicological assessments on an ongoing as needed basis, and a qualification process for unknowns. And for the container closure system and SUSs, one must ensure it is possible to fully characterize and understand the materials of construction and their associated extractables. This may take the form of controlled extraction studies, simulation/migration studies, and drug-device interaction studies.

 At those production steps where accumulation of leachables is expected, sampling should be performed and leachables monitored. This should become part of any process qualification.

	The profile from different sampling points may be mapped, with the leachables profiles that have been generated after the last filling step. This is to assure that no leachables have been accumulated over the process line. In some cases, target analysis by use of semi-validated methods must be performed to assure that any expected leachables of toxicological concerns have been purified from the process stream.

	The sampling strategy depends on the individual biopharmaceutical process and how trustable the single-use components are chemically characterized. A poorly characterized chemical profile in a SUS component poses a higher risk to the process.

 In biomanufacturing processes, people look for the presence of E&Ls through the entire process from the earliest biomanufacturing steps to the very end of the process and all stages in between. Testing is done at multiple stages of biomanufacturing in order to understand how each individual component of that process will have an effect on E&L. In the case of the bioreactor, it is very far upstream in the process--very far from the patient--so it typically has a very minimal impact on safety. E&L should be understood as a function of risk, which needs to be characterized and controlled throughout the entire process.

	Regarding what can be done in real-time, there is a lot of innovation going on right now where people have the ability to sample a product throughout bioproduction. They can do different tests that allow one to make modifications based on those results, whether it be a simple total organic carbon content measurement or even just a sampling where one would perform some of the traditional methods used in E&Ls testing such as gas chromatography-mass spectrometry (GC-MS) tests or other analytical techniques. Again, it goes back to the risk characterization and determining what matters most when evaluating E&L impact. There is no one-size-fits-all approach. 

 How much of a concern is E&L in biomanufacturing compared to traditional small-molecule pharmaceuticals, for example?

 As noted previously, the single-use biomanufacturing process that commonly is used for production of large-molecule products increases the amount of direct contact of the process stream with polymeric and elastomeric component materials. That results in a higher degree of overall risk for leachables being introduced to the product, which may interact directly with the product or which may potentially pose a safety concern directly to the patient.

	Leachables often present a greater concern in general for biologic and other large protein molecules compared to small-molecule formulations, and this greater concern for large molecules applies as well to leachables that may be introduced from the biomanufacturing process.

	There are multiple reasons why large molecules may present a greater concern for leachables. The large size and complex, conformational structure of proteins provides a higher surface area and an increased number of potential active sites where leachable compounds (organic or inorganic) may interact with the protein.

	Large-molecule formulations often include solubilizing agents such as polysorbates, which may hasten migration of organic leachables by improving the solubility of such organic compounds in an otherwise typically aqueous medium. The amphiphilic nature of proteins themselves also may hasten the solubilization of organic leachables.

 In all production lines where stainless steel has been used, the leaching problem is a lower risk compared to processes where single-use technology has been used. Large-molecule formulations are composed of more complex matrices, which may contain surfactants such as polysorbate or poloxamer or related substances that could easily interact with plastic surfaces.

	It is often very challenging, or not possible, to discover leachables at trace levels in that “chemical cocktail” by screening methods (not targeted analysis). Complex sample preparations are generally required for biological formulations and there can be issues associated with the recoveries of leachables in the biological matrix.

	Proteins could easily capture or bind with lower mass leachables and they do not release them in solution again. Thus, these leachables would run out of the analytical window. In small-molecule manufacturing, discovering of impurities is much more easily performed.

 Because a biologic is a larger molecule and has multiple reaction sites, the presence of other chemical constituents like E&L will have a higher potential for reacting with the biologic, and having some impacts either on the efficacy of the product or on safety to the patient.

	The way biologics are made--using single-use systems and having various polymeric systems exposed throughout the entire process--is not as common in small-molecule manufacturing. For small molecules, the manufacturing process traditionally uses glass or stainless-steel reactors. By having fewer polymers exposed, the source of E&L is reduced dramatically.

	E&L is a much higher concern for all of the reasons that I just referred to and needs to be characterized accordingly when considering the manufacturing of biologics. The best way to do that is through a comprehensive risk management process followed with appropriate chemical characterization. The trend in recently developed regulatory guidelines reinforces this approach.

A Cytotoxic Leachable Compound From Single-Use Bioprocess Equipment That Causes Poor Cell Growth Performance

 online, DOI: 10.1002/btpr.1869, Feb. 5, 2014.

In a roundtable Q&A with biopharma executives, the vulnerability and challenges of dealing with extractables and leachables in single-use bioreactor bags are explored.