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So, what’s not in your standard operating procedure? Documenting details can prevent headaches associated with method transfers between laboratories.
Dwight R. Stoll1 and Tony Taylor2, 1LC Troubleshooting Editor, 2Crawford Scientific, Strathaven, Scotland, UK
So, what’s not in your standard operating procedure? Documenting details can prevent headaches associated with method transfers between laboratories.
About a year ago, I began considering the handoff of the “LC Troubleshooting” column from John Dolan. As I started discussing potential topics to address with people in the liquid chromatography (LC) community, the topic that came up most frequently was method transfer, loosely defined. And based on further discussion recently, it has become clear to me that challenges associated with this topic are only going to become more prominent in the near future. Also, the landscape around this topic is highly varied, and detail and nuance are extremely important. This means that the topic cannot be adequately addressed in a single instalment of “LC Troubleshooting”. Indeed, I expect to see the topic addressed frequently in this magazine in the coming months and years, both by myself and other experts. With this context in mind, I am viewing this month’s column as setting the stage for subsequent discussions-giving some background on how we have come to this point, and discussing the scope and nature of the problem by way of example. For this purpose, I have asked Tony Taylor of Crawford Scientific and LCGC’s ChromAcademy to join me this month. Tony’s deep experience in chromatography training and consultation gives him a rich and unique perspective to draw upon that cuts across both manufacturers (that is, instrument and consumables vendors) and consumers (for example, scientists at pharmaceutical companies) of chromatography. We hope you enjoy the discussion. – Dwight Stoll
What Exactly Do We Mean By Method Transfer?
In today’s world of method development and life cycle management, the idea of method transfer can mean different things to different people. For example, with all of the recent changes in particle and column technology there has been increasing discussion about strategies for “transferring” a method based on high performance liquid chromatography (HPLC) technology (for example, 5-μm fully porous particles) to an ultrahighâpressure liquid chromatography (UHPLC) platform (for example, subâ2âµm superficially porous particles) (1,2). This concept is sometimes referred to as method translation. There is no question that this is a very important topic, but it is beyond the scope of what we will address here. Instead, the focus of our discussion is on scenarios that involve first the development of a method in one laboratory (typically, called the originator laboratory or the transferring or sending laboratory), and second, the implementation of that method in a physically different laboratory (typically, called the receiving laboratory). In the best case scenario, the receiving laboratory would be able to reproduce results obtained by the originator laboratory to within the uncertainty of the measurement. However, there are many factors that can lead down a path where this ability to reproduce results cannot be demonstrated. In this instalment, we highlight some of these factors and suggest ways to avoid
How Did We Get Here?
We seriously doubt that the generation of scientists that developed HPLC in the 1960s and 1970s ever imagined that a method developed on an instrument in Boston might be transferred to a laboratory in Mumbai with the expectation that the same results would be obtained in both locations-at least not in the early days. In those days, most methods were executed for years within the same company that developed the method, if not the same building. However, since the early 2000s it has become increasingly common for a method developed in one location to be executed in a different location for the remainder of the lifetime of the method. A significant driver for this has been the trend towards outsourcing of manufacturing and even method development in certain industries and application spaces (3). In some cases, the fact that the location is different is the least of concerns-the originator and receiving laboratories may be in different companies, running instrumentation from different vendors, and using reagents from different suppliers. It is not hard to imagine how this situation can quickly lead to problems. There is not too much literature on the topic of method transfer before about 2010. However, in just the past year or so several substantive articles on the topic have appeared in chromatography, pharmaceutical, and life science journals, which reflects the increasing importance of this topic to the community (4–8).
How Does Method Transfer Typically Work?
One needs to carefully consider the “stage of development” at which transfer occurs. These may include preclinical methods that are transferred to a different laboratory for analysis of clinical samples, methods initially developed with a focus on an active pharmaceutical ingredient (API) that subsequently need to be implemented in stability studies, and finally methods that have been developed and validated in the originator laboratory but then need to be implemented in the receiving laboratory for routine use. Of course, some method adjustments may be required in these situations. However, while transfer of methods that have been fully validated and in routine use should be straightforward, these can be the cause of as much consternation as those in which some adjustments are expected from the start.
Further, the regulatory requirements of the receiving laboratory must be borne in mind if regulatory submission or the use of methods for product quality control (QC) is intended. Where regulatory requirements differ, this difference should be a principal concern during method transfer.
When transferring methods, it is typical to develop a method transfer protocol, which includes details of the method (including sample preparation and data handling), the test articles that will be used including the number required to give statistically meaningful results, and any special requirements (forced degradation lots, placebo lots formulated with known impurities, and so forth), any special sample handling requirements, the number and type of tests being undertaken, and, critically, the acceptance criteria for a successful transfer.
The transfer protocol is often primarily focused on the acceptance criteria to use when considering the resulting data from both laboratories, and there are many different ways in which the accuracy, precision, sensitivity, ruggedness, and robustness of the methods may be assessed, and the statistical analysis of the data that will highlight any interlaboratory variability in the results. Most regulatory guidance will refer to “acceptable” performance evaluation, but United States Pharmacopeia (USP) Chapter <1224> “Transfer of Analytical Procedures” (9) does give more helpful guidance. Helpful testing regimes, statistical data analysis approaches, and acceptance criteria have been proposed by several authors. A few that are particularly noteworthy are the procedures and equivalence tests recommended by the International Society for Pharmaceutical Engineering (ISPE) (10), a further treatment of these procedures to reduce the required effort and further develop acceptance criteria by Kaminski, Schepers, and colleagues (11), and the use of ratio-based testing using methods such as Bland-Altman plots with limits of agreement (12).
All of the above being said, the devil is always in the details, and it is invariably true that increasing the level of detail supplied to the receiving laboratory will always improve the chances of a successful method transfer (7,9). This additional information includes the detail supplied regarding the actual analytical method, where the standard operating procedure (SOP) or standard method of test (SMT) is usually not enough to capture the “vagaries” of the originator laboratory. It is far better, where possible, to include details on the method development (even in a short summary document), any validation protocols and reports as well as the control documents for methods in routine use that will identify trends and patterns in calibration samples, QC samples, and the results from “live” test articles.
So, What’s Not in Your SOP?
In discussing method transfer with scientists on both the originator and receiving ends of a method, we have heard very different perspectives. From the receiving end we frequently hear scientists exclaim, “We did exactly what they told us to, and the method didn’t work!” And, from the originator we hear, “They didn’t do what we told them to do!” Upon further discussion it becomes clear that both parties are partially right, and the two views can be reconciled by realizing that not enough detail was specified in the method transfer protocol. This is akin to the oft-cited idea in computer science that computers only do what we tell them to do:
“The Analytical Engine has no pretentions whatever to originate anything. It can do whatever we know how to order it to perform.” - Ada Lovelace, Notes on the Sketch of the Analytical Engine, 1842 (13)
So, it is also with an analytical method-if we do not specify all of the details necessary to reproduce the results of the analysis, we should not expect them to be reproduced faithfully. However, specifying all of the details is often not a trivial task. For example, details relevant to the preparation of buffered eluents may be such a well-established part of the culture of the originator laboratory that they are not included in the method. The originator laboratory may prepare a 0.1% solution of trifluoroacetic acid in water on a w/w basis, while the receiving lab may prepare the solution on a v/v basis-because that is the way they usually do it. In many cases this difference may not cause a problem, but we have certainly seen situations where it can cause a problem with the method transfer. So, this brings us to the following question: What’s not in your SOP (that should be)?
The following list is a set of examples of such details that have caused problems in our own work, or have come up in discussion with scientists from different companies. It most certainly is not an exhaustive list of things to focus on. Rather we hope it motivates readers to include more detail in methods that will be transferred-again, with the aim to increase the likelihood of a successful method transfer.
Vials and Septa: As chromatographers, we would prefer to focus our efforts on optimizing the chromatography itself, rather than having to worry about such details as the make and model of the sample container. However, these details can be very important indeed (3). Different types and grades of septa have different extractable profiles and can affect injection carryover, because some injector designs rely on the vial septum to wipe down the outer surface of the injector needle. The vial material itself (for example, glass versus plastic) and how the material is treated at the time of manufacture (for example, washing or deactivation of glass) are also important. These details can affect adsorption of certain analytes, particularly at low concentration, and contamination at the time of manufacture can lead to the appearance of “ghost peaks” (14–16) in blank samples later on. Thus, where possible the same make, model, and grade of vial and septum should be used in both the originator and receiving laboratories.
Detector Variables and Settings: There are many detection variables that can affect the success of method transfer. One that has become more important in recent years is the data acquisition rate. As the resolving power and speed of LC have improved with advances in instrument and column technology, peaks have decreased in width on average, both in terms of time and volume. Methods that produce peaks that are very narrow in time require the detector signal to be recorded more frequently compared to past experience. Acquisition that is too slow can compromise resolution by making peaks appear broader than they are upon elution from the column. On the other hand, using the highest acquisition rate available all the time can lead to signals that are too noisy. Most modern ultraviolet (UV) absorbance detectors support these acquisition rates very nicely, and there is good guidance in the literature on what acquisition rate should be used for a particular peak width (17). Nevertheless, we need to be diligent about making sure this setting is specified in the method transfer protocol. Also important are factors that affect detection sensitivity and signal-to-noise ratio. In the case of UV absorbance detection, these include reference wavelength, detection bandwidth, and detection pathlength. Again, these parameters should all be clearly specified to increase the likelihood of a successful method transfer.
Column Conditioning: One of the aspects of chromatography that makes it so powerful as an analytical tool is that very subtle changes in analyte or stationary-phase chemistry can result in selectivity differences big enough to resolve two peaks easily. However, this aspect has a dark side too. Subtle changes in column chemistry as the column ages over its lifetime can lead to changes in selectivity that can turn good resolution into bad resolution. In other words, this type of change can cause a method to drift out of specification or fail system suitability tests. Although it would be convenient if a column worked as it should with the very first injections out of the box, this is often not the case. We have first-hand experience with this and have heard several anecdotes about situations where a well-defined column conditioning step was required to “break-in” a column before using it for the intended analysis. This step may simply involve exposure to the mobile phase for a specified number of hours (18) (or even days in our experience!), or perhaps several injections of the target analytical samples before using the column to collect data on samples of interest. Sometimes this process results in in situ modification of the stationary phase by constituents of injected samples; indeed, this is why John Dolan has recommended for years that columns be dedicated to specific projects (19). On average, these problems are less serious today than they were in the early days of HPLC because column technologies have improved dramatically, but they still deserve attention.
Connections: Variation in Tubing Length and Diameter: Most modern LC instruments provide users with opportunities to change the plumbing of the system, particularly the connections that are made between the pump, injector, column, and detector. This capability, too, can be both a blessing and a curse. For example, in recent years there has been a lot of discussion about reducing the diameter of connecting tubing to minimize peak dispersion outside of the column (17). Minimizing extracolumn peak dispersion is very important from a conceptual point of view, but we have to be careful to make sure we do not end up operating too close to performance margins, and allow some room for variation in the tubing itself. The pressure drop across a piece of tubing under laminar flow conditions scales with the fourth power of the tube radius, so even very small variations that might arise from different manufacturing batches of tubing, or between different manufacturers, can lead to a significant change in system pressure, and could even exceed the pressure limit of the pumping system (20).
As the chromatography community acquires more and more experience with method transfer, best practices will emerge and the frequency of good experiences with method transfer should increase as a result. Indeed, these best practices are beginning to emerge as experts publish guidance that can be applied broadly across different industries and companies (3–5,8). The following short list describes best practices that will support successful transfer of chromatographic methods.
Document the Details: When in doubt, include more details in a method transfer protocol, even if they seem irrelevant at the time. Even though “one-to-one” implementation of the method in the receiving laboratory using equipment and materials identical to those in the originator laboratory is not always possible for a variety of reasons, including more detail in the transfer protocol at least makes it more likely from the outset. Examples include specifying the source of vials, pipette tips, centrifuge tubes, and wellplates. Also, including the many details associated with complex operations-for example, ionization conditions for electrospray mass spectrometry-could save a tremendous amount of time and effort in the transfer process.
Use Available Data and Tools to Prevent Problems Before They Occur: As more data and software tools relevant to the method transfer process become available, there will be more opportunity to anticipate problems before they occur. For example, the recent publication by Ahmad, Blasko, and colleagues (6) describes extensive comparative data on five state-of-the-art UHPLC systems, ranging from gradient delay volume of the pumps to the time required for thermal equilibration of samples in thermostated autosamplers. Especially when it is known that the method will be transferred to a receiving laboratory with different HPLC equipment, these comparative data may be useful in anticipating differences in the performance of the method in the originator and receiving laboratories. Also, instrument control software is becoming more sophisticated in ways that support method transfer. For example, some systems now have the ability to mimic the behaviour of systems from other manufacturers. In principle this approach can simplify method transfer in cases where the originator and receiving laboratories are using systems from different manufacturers.
Communicate Early and Often: The value of communication in the method transfer process cannot be overstated. Hilhorst and coworkers, writing about the transfer process from the perspective of a contract research organization (CRO) (3), emphasized the importance of mutual respect for the work of the originator and receiving laboratories. They also suggested that training receiving laboratory scientists in the originator laboratory is very helpful in the long run, even if it is expensive on the front end of the process. If travel to the receiving laboratory is not possible, then webâbased communication tools are very helpful in the process of identifying problems during the facile processing of samples and instrument setup. We can perhaps imagine a day in the not too distant future where tools such as the GoPro camera or Google Glass can be used to truly “follow” workers at a different site undertaking an analysis to spot “obvious” issues that are otherwise difficult and time consuming to solve using only email and phone conversations.
Mind the Volumes: One of the most frequently cited causes of problems in HPLC method transfers is differences in volumes of different parts of the instrument in the originator and receiving laboratories. As HPLC equipment has evolved over the past three decades, gradient delay and extracolumn volumes have changed significantly, and differences in these volumes can have significant effects on method performance. It is fair to say that these effects are well understood at this point, with detailed guidance available to help understand the challenges and potential solutions (8,21). Now, the challenge is to leverage this knowledge in practice, and to anticipate potential problems before they occur.
Minimize Variation Where Possible: The circumstances of some method transfers will prevent one-to-one transfer for reasons beyond the control of the scientists involved (for example, HPLC equipment from different vendors in the two laboratories). For other aspects there is more control, however, and for these, every effort should be made to minimize variation between laboratories. For example, one laboratory may routinely filter their mobile phases, whereas the other laboratory may not (see the February 2017 “LC Troubleshooting” for details on this point in particular ). This case is an example of a detail that is highly dependent on laboratory culture and habits, and may not normally be specified in the method. Also, the quality of reagents used in a method can significantly impact results. For example, the use of lowâgrade mobileâphase additives can lead to unexplained “ghost peaks,” particularly for methods involving gradient elution. If these ghost peaks show up on analyses of blank samples, they will have to be investigated; where possible it is best to simply avoid these problems by using reagents of the same quality in both laboratories. Again, as discussed, include as many details as possible, even if they do not seem important at the time (23).
Where Do We Go From Here?
The topic of method transfer encompasses a vast space that includes many concepts in laboratory practice and skill, chromatography, and instrumentation, and is affected by a variety of organizations and people including instrument, consumable, and reagent vendors, chromatography training organizations, originator laboratories, CROs, and contract manufacturing organizations (CMOs). If the last two years are any indication, we will see this topic being addressed in the analytical and chromatography literature more frequently in the near future. This literature promises to provide both guidance (for example, see reference 4) and data (for example, see reference 6) that users can draw on to increase the likelihood of a successful method transfer from, or to, their laboratories. Out of curiosity we searched the technical programmes from the 2016 meetings of AAPS and Pittcon and found very little evidence of discussion of the method transfer topic in an explicit and organized way. Perhaps going forwards, it would be helpful to organize one or more technical sessions at Pittcon and other conferences that would bring together parties affected by method transfer to discuss challenges and develop a shared sense for how to move forwards as a community. In the coming months, we and others will be tackling more specific aspects of method transfer in LCGC.
We thank Todd Maloney, Kelly Zhang, Peter Johnson, Lianjia Ma, and Bill Long for their helpful discussions leading to the preparation of this instalment.
Tony Taylor is the technical director of Crawford Scientific and ChromAcademy. He comes from a pharmaceutical background and has many years of research and development experience in small molecule analysis and bioanalysis using LC, GC, and hyphenated MS techniques.
Dwight Stoll is the editor of “LC Troubleshooting”. Stoll is an associate professor and co-chair of chemistry at Gustavus Adolphus College in St. Peter, Minnesota, USA. His primary research focus is on the development of 2D-LC for both targeted and untargeted analyses. He is also a member of LCGC’s editorial advisory board. Direct correspondence to: LCGCedit@ubm.com