January 17, 2024 PAO-12-23-CL-09
Liquid chromatography (LC) involves the separation of compounds on the basis of their physicochemical properties. A liquid solution containing the target molecule and impurities is passed through a tubular column packed with a chromatographic medium. Typically, both the target molecule and the impurities interact with the stationary phase in the column, but differences in those interactions cause them to have different elution times in the column, allowing for separation of the target from the impurities at different time points during elution.
Several different types of chromatography — such as affinity, ion-exchange, or size exclusion — can be employed. Additionally, within each of these chromatography modes, many different resin chemistries are available. The specific resin used in a given chromatography step will be determined by the nature of the target molecule being purified and the goal of the purification step. In many cases, two or more chromatography steps using different conditions are needed to reduce the content of various impurities to the necessary levels.
HPLC refers to high-performance (alternatively, high-pressure) liquid chromatography and involves passing the liquid through a very thin column at high pressure to achieve greater separation. UPLC refers to ultra-high-performance LC and involves even higher pressures and narrower tubing.
HPLC is the most widely used liquid chromatography technique throughout the biopharmaceutical industry, leveraged for applications across all stages of drug development from discovery through commercial manufacturing and product release. As an analytical method, HPLC is used during discovery to help identify novel compounds with interesting bioactivities, in process development to determine optimum reaction conditions, and on the manufacturing plant floor to monitor processes and ensure that they are performing appropriately.
HPLC is also an important QC method for confirming that final products meet specifications prior to release. It is important to note that method development for quantitative HPLC analysis (to determine the amount of a substance in a mixture) is more burdensome than method development for qualitative HPLC analysis (to determine if a substance is present in a mixture).
Preparative HPLC, meanwhile, is deployed for the purification of intermediates and drug substances. Small-scale processes are developed using miniaturized equipment, and microgram quantities are then transferred to larger-scale columns in which kilograms of material are purified.
Ultimately, HPLC presents an efficient means of separating molecules. Elution of the column is followed by analysis using some type of detector to generate a chromatogram. Common detection techniques include ultraviolet-visible (UV-Vis), electrochemical, and fluorescence detection; mass spectrometry is also increasingly used today. Other methods include refractive index, evaporative light scattering, and charged aerosol detection.
If a molecule does not have properties that allow for detection by these techniques, HPLC is not an appropriate analytical method. Highly ionic or polar molecules (e.g., magnesium, potassium chloride, and lithium) can also present an issue for HPLC: they immediately flush off a reversed-phase column and bind very tightly to hydrophilic interaction columns (HILIC). If an HILIC method is found to work, it would be expensive because HILIC columns cannot be reused as many times as other HPLC columns.
Most drugs based on small organic molecules, fortunately, are amenable to separation/purification using HPLC and detection via UV-Vis at wavelengths from 190 to 400 nm. Even for drugs where common techniques are not applicable, mass spectrometry, although expensive, can be an effective alternative means of detection. Nuclear magnetic resonance spectroscopy is yet another option, but it is the most expensive of detection techniques.
Timelines for method development vary significantly depending on the nature of the mixtures to be analyzed. For a simple mixture containing a molecule that falls within a class of compounds for which HPLC behavior is well understood, the development of an effective method may be achieved in a day or so. Challenges arise for molecules in new, uncharacterized drug classes or those that are sensitive under typical HPLC conditions, such as those that readily oxidize, hydrolyze, de-alkylate, or otherwise degrade. Highly complex mixtures also create difficulties. In these cases, successful method development may take days or even weeks.
The goal is always a robust and reproducible method that provides the same results regardless of the technician, the instrument, or the laboratory in which it is performed. Cost-effectiveness is a secondary but still important consideration. Reversed-phase HPLC methods (polar solutions are used to pass the mixture onto and through a nonpolar stationary phase) are optimal, as this type of chromatographic medium is one of the least expensive. Ion-exchange and hydrophobic-interaction media are more expensive and often also require the use of dedicated analytical instruments, which further drives up costs.
The level of separation required for different HPLC methods depends greatly on the purpose for which the method is intended. For process monitoring during process development, it may not be necessary to clearly separate every molecule in the reaction mixture — the key might be to ensure that the product peak is well separated from all other peaks. Similarly, an assay for confirmation that the drug substance quantity in a final drug formulation meets specifications only requires clear separation of the drug substance. A method developed for impurity analysis, however, must provide clear separation of each peak in order to allow for identification and potential quantification of those undesired compounds.
The timing of HPLC method development also depends on the stage of the development process and the type of drug product. Companies developing truly novel compounds should ideally initiate method development once the active pharmaceutical ingredient (API) has been confirmed. It is essential to have a robust and reproducible HPLC method for detection of the API. It is equally important to have effective HPLC methods ready for use during process and formulation development, including (for the latter) specific methods for evaluating the results of drug–excipient compatibility studies. Additionally, validated methods are required for product release and are often developed in parallel.
Companies developing generic drugs may have access to pre-existing methods, such as those published by the U.S. Pharmacopeia (USP). In those instances, some optimization may be required, but it is generally preferable to leverage validated USP methods whenever possible.
Understanding how the chemical structure of a molecule impacts its behavior on different chromatographic media under different conditions can help reduce method development time. For instance, highly polar molecules that contain many electron-rich or electronegative atoms will bind tightly to polar stationary phases but flow directly out of a reversed-phase (nonpolar) column. Functional groups on the molecule must also be considered. Highly ionizable substituents will behave differently at different pH levels, while others might form ion pairs.
Evaluating the structures of the target molecule and other compounds in the mixture allows for the identification of chemical features that can then be exploited to enable separation. It may be possible to ionize, protonate, deprotonate, or otherwise change the nature of the target molecule to sufficiently differentiate it from the other compounds during HPLC. For some highly similar compounds that must be separated, a change in temperature, pressure, or mobile phase gradient may be needed. This type of knowledge also makes it possible to identify mobile phases that should be avoided because they may react with the molecule and lead to the formation of impurities that are not in the original sample.
Previous experience with a wide range of molecules and mixtures is thus invaluable at this stage and can help to more quickly reduce the choices of mobile and stationary phases to explore. Today, that can include the use of artificial intelligence–based programs that evaluate different attributes of a target molecule, such as its structure and polarity, to predict potential degradation impurities and optimal HPLC solutions.
Successful HPLC method development requires developing a working relationship with the equipment and detection instruments. Columns and detectors need to be properly cleaned. To extend the lifetime of a packed HPLC column and ensure robust and reproducible performance, it must be rinsed with the appropriate solutions following each run. For columns used by multiple different technicians in a lab, it is critical to pay attention to what purifications are being done by whom and when so that critical cleaning can be performed properly. In addition, it is essential to clean other components of the system, including the seals, needle, and so on, to avoid residue buildup.
There are clear benefits to taking a holistic view of method development. It is often common during method development to get bogged down with a specific issue and the many small changes that can potentially be made to resolve it. For instance, it may be difficult to fully separate two very close peaks. If making some obvious small change does not help, it is often useful to take a broader perspective. Perhaps changing the temperature or even changing the instrument or shortening the tubing length may help.
When performing HPLC runs, it is equally important to avoid overloading columns, which can lead to the formation of a fronting peak and exceed the capability of the detector (i.e., pegging the detector). This can prove disastrous for quantification methods because the detector can no longer function properly and will report values that are too low.
Knowledge of the specific resins being used for HPLC is also essential. Even within the same resin chemistry, there can be important differences in HPLC columns obtained from different manufacturers. For instance, silanol groups may not be capped the same way in all reversed-phase columns, which can have a dramatic impact on method performance.
As a result, securing the supply chain for HPLC materials is critical to successful method development. It is essential to have access to the same column (and sometimes from the same manufacturing run) throughout the life of the project. It is also ideal to have a reliable supplier of buffering chemicals, although as long as the salt, base, or acid is of the same purity, the supplier generally does not matter (unless there are new impurities that influence the separation properties).
Wherever possible, it is advisable to run methods on the same instrument, even though the methods once developed are intended to provide equivalent results on multiple similar instruments. If switching to a different instrument is necessary and some issues are observed, regulatory agencies and USP methods allow certain minimal adjustments to help overcome such differences in performance. They include raising/lowering the temperature a few degrees, slightly shortening or lengthening the column, and slightly changing the compositional percentages in the mobile phase.
Successful HPLC method development requires effective collaboration among all users, drawing upon previous experience, deep chemistry and analytical knowledge, and appropriate respect for and care of equipment and materials.
For methods that will be used in process development, close interaction with the R&D scientists is essential to ensure that the resulting methods will be appropriate for their intended use. For methods intended for use in QC applications, two-way communication between R&D and QC personnel helps to ensure that methods are appropriate for use in the QC environment: the equipment is available, the methods are not overly complex, and the conditions can be implemented. Working with formulation scientists is equally important, as some formulation excipients can damage the type of column needed for HPLC analysis of the drug substance, and excipients might react with the API under the analysis conditions. In other cases, a particular excipient may be incredibly difficult to separate from the drug substance.
For CDMOs, collaboration with clients is incredibly important. In many cases, drug developers have extensive prior knowledge and experience that can be invaluable when developing HPLC methods. That is particularly true if the client wants the CDMO to transfer in the client’s method and verify and validate it. If issues are identified with the method, that prior knowledge can help determine how to resolve them, or, in some cases, that the issue is not relevant to the required performance of the method.
As a CDMO dedicated to supporting small molecule drug manufacturers, Mikart has solidified its reputation by investing in state-of-the-art R&D laboratories and fostering a culture of hands-on involvement. At the heart of this innovative approach is Mikart's Analytical R&D Director, who not only oversees operations but actively participates in laboratory work. This unique approach fosters an environment of open communication, knowledge sharing, innovation, and open-mindedness.
Mikart's commitment to innovation extends beyond the traditional boundaries of contract services. When transferring a client's method into their facilities, Mikart's teams approach it with a proactive mindset. If challenges or questions arise during method validation, they don't just identify issues — they collaborate with the client to propose comprehensive plans of action. This proactive, problem-solving approach aims not only to address immediate concerns but to continually refine and enhance methods, ultimately delivering improved performance for both Mikart and their valued clients.
Moreover, Mikart remains at the forefront of industry advancements. The organization is dedicated to staying current with the ever-evolving landscape of analytical science. By actively monitoring and incorporating the latest developments and best practices in the field, Mikart ensures that their clients benefit from cutting-edge solutions and optimized processes. This unwavering commitment to innovation and flexibility exemplifies Mikart's dedication to exceeding industry standards and consistently delivering exceptional value to their partners in the pharmaceutical arena.
Ransom Jones is an accomplished analytical development scientist with a robust background in chemistry. His contributions to the scientific community, highlighted by publications in Chemistry Europe in Medicinal Chemistry & Drug Discovery, showcase his expertise. With a foundation in process chemistry and a focus on scale-up methods, synthetic pathways, and purification techniques, Ransom excels in drug development. His graduate training specialized in medicinal chemistry, particularly in synthesizing antiviral compounds for various viruses. Currently, at Mikart, he applies his skills in HPLC Method Development, Validations/Verifications, and Analytical Development. Ransom graduated Valedictorian with both a BS in pharmaceutical sciences and an MS in pharmaceutical and biomedical sciences from the University of Georgia.