As chromatography technologies change, our approaches to using them in ways that value reliability, as well as the ways we should approach troubleshooting and fixing problems, should also change. In this installment, I continue the discussion started last month about how some specific troubleshooting topics have evolved over time, this time focusing on mobile phase degassing. Understanding how approaches to degassing have evolved is helpful for troubleshooting problems with pumps and detection, designing reliability into new methods, and when considering updates to legacy methods that have been in use for decades.

In recent months, I’ve been hearing from several readers of this column that they’ve appreciated the more educational, “back-to-basics” flavor of some of the “LC Troubleshooting” installments in the last year. So, last month I decided to dip a little further into that theme and provide some perspective on the evolution of various troubleshooting topics over the last few decades. My view is that this kind of perspective is particularly valuable to those who are relatively new to the field. There are some aspects of “the way we do things” that may seem peculiar on the surface, but are in fact very important to the reliability of high-performing liquid chromatography (LC) methods. On the other hand, some aspects of certain methods and ways of doing things are simply unnecessary in 2023 because LC technology has evolved in such ways that the old tricks aren’t needed anymore. Sometimes implementing the old tricks with new technology – though they provide no benefit – doesn’t do any harm, but they do add cost to analyses because they take time and resources to implement. Thus, we really ought to let them go if they are not adding any value to the method. In other words, let’s be smart about the methods we deploy. If we can’t come up with a better explanation for why something about the method is the way it is, then it’s time to let it go. In this installment, I will discuss the evolution of degassing in liquid chromatography. In general, degassing has become much more convenient, to the extent that most users probably rarely think about it anymore. However, some of the older techniques are still useful, and even with modern techniques it is useful to have a broad sense for how things have changed, as this can impact proper instrument operation when working with different generations of equipment. Readers interested in learning more details about the degassing topic are referred to previous installments of “LC Troubleshooting” (1,2), Dolan’s book on LC troubleshooting (3), and a very old, but very rich, paper by Bakalyar and coworkers (4).

In most applications, the biggest problem with gas bubbles inside of an LC system is that pumps generally don’t deal with the bubbles very well (see the last section below for other problems that can be significant in some applications). In the worst cases, a gas bubble can cause one or both of the check valves in a high-pressure pump to fail, causing a highly erratic flow or no flow at all. Where, then, do these bubbles come from? Although there are multiple mechanisms that can lead to bubble formation, the most practically relevant one in LC is the situation where two solvents are brought together to make a mixture (here, the mobile phase) that has a lower gas solubility than either of the individual solvents alone. This is most problematic with pumps that use the “low-pressure mixing” design. For a refresher on the differences between low-pressure and high-pressure mixing designs used in LC pumps, readers are referred to previous articles in this magazine (5). In the case of low-pressure mixing, the individual solvent components are brought together under nominally atmospheric pressure conditions before a high-pressure pump provides the force needed to push the mixture through the rest of the LC system.

An example is valuable for illustrating the point here. The solubilities of oxygen gas at room temperature and atmospheric pressure in water or ethanol are about 0.3 × 10

, respectively (4) (albeit on a mole fraction basis, meaning moles of oxygen relative to the total moles of oxygen and solvent). In other words, when saturated with oxygen, ethanol carries about 20 times more oxygen dissolved in the solvent compared to water. If we mix the two solvents in equal parts, then the amount of oxygen present in the mixture initially will be about 3.0 × 10

 (mole fraction). However, the solubility of oxygen in a 0.5 (mole fraction) mixture of water and ethanol is only about 1.8 × 10

 (mole fraction). This difference of 1.2 × 10

 is the amount of oxygen in excess of the carrying capacity of the mixed solvent at saturation, and this excess will manifest in the formation of significant bubbles. In a low-pressure mixing pump, these bubbles will be drawn into the pump, and may cause failure of one or both check valves. So, in this example, avoiding the problem requires that the concentration of gas dissolved in each of the individual solvents is decreased prior to mixing such that the gas concentration in the mixed solvent (mobile phase) is lower than the solubility limit of the gas in that solvent. If this is achieved, then bubble formation will not be nearly as serious. A different way of thinking about the same problem is to consider the volume of gas that will be evolved as bubbles upon mixing of two solvents. Figure 1 shows data along these lines from the work of Bakalyar and coworkers (who physically measured the volumes of bubbles formed using an apparatus for trapping the bubbles) for mixtures of methanol or acetonitrile with water (4). We see that the volumes can be quite remarkable – 60 μL of gas per milliliter of mixed solvent! – and that these volumes roughly maximize around 50:50 mixtures of the organic solvent and water.

The situation with pumps that use a high-pressure mixing design is quite different. In this case the mixing point for the mobile phase components is downstream from the high-pressure pump heads, and the pressure at which the mixing occurs will be nominally the same as the column inlet pressure (that is, well above atmospheric pressure). Under these conditions the gas solubility in the mobile phase will be much higher and bubbles will not form while the liquid is under pressure. However, as the pressure drops toward atmospheric pressure at detector, bubbles may form when the gas concentration reaches the solubility limit in the mixed solvent. If bubbles form in an optical flow cell (for example, ultraviolet-visible (UV-vis) or fluorescence), this can lead to unstable baselines, including spiking patterns.

Survey of Degassing Techniques, Old and New

Including older discussions of degassing techniques used in LC, the list includes: heating, sparging, refluxing, sonication, offline vacuum degassing, and inline vacuum degassing. I personally am not aware of anyone currently routinely using heating or refluxing for degassing, and I won’t discuss those approaches further here.

I like to think of sparging as a technique that “scrubs” dissolved gases from a liquid. The principal fact in play here is that helium is much less soluble in LC solvents than other gases, including oxygen and nitrogen. In methanol, which is particularly problematic as discussed above, the solubilities of these three gases at room temperature and atmospheric pressure are about 0.7 × 10

 (mole fraction), respectively (4). To use the sparging approach in practice, helium is deliberately bubbled into a LC solvent. Other gases dissolved in the liquid diffuse into the helium bubbles and are carried out of the liquid as the helium bubbles rise to the surface. After an initial scrubbing period (15 minutes of sparging will remove about 80% of the oxygen dissolved in methanol [6]), the “gas-free” solvent can be maintained on the instrument with a very low flow of helium into the solvent bottle. Helium sparging has fallen out of favor due to the introduction of other approaches (mostly inline degassing, discussed below), practical inconvenience (nobody wants to deal with compressed gas cylinders if they don’t have to), and the cost of helium, meaning it is not commonly used today. That said, I do still have LC instruments in my laboratory that have the necessary plumbing and sparging stones to support this approach.

A very simple approach to degassing is to simply place a bottle of solvent to be used on the LC instrument in an ultrasonic bath for several minutes before use. Unfortunately, this approach is not very effective; fifteen minutes of sonication will only remove about 30% of the dissolved oxygen dissolve in methanol (6).

In the offline vacuum degassing approach, a vacuum is applied to a bottle containing the solvent that will ultimately be used on the LC. The most convenient and safe source of vacuum would be a house vacuum system, as found in many laboratories. The fundamental idea here is that reducing the pressure in the bottle decreases the solubility of gases dissolved in liquid, and any gas present above the solubility limit will spontaneously bubble out of the liquid. The vacuum can be applied using a sidearm type of flask with a hosebarb connection, or a plastic bottletop adapter with a hosebarb fitting that can be used to connect to the bottle to the vacuum source. When using this approach, great care should be taken to ensure that the container placed under vacuum is not physically compromised with cracks or other defects, as this could lead to a dangerous implosion of the container. Using a safety shield when the bottle is under vacuum is a good idea. Users should also be careful to use an explosion-proof vacuum pump if using a pump as the vacuum source, particularly when working with solvents that produce explosive vapors.

In my laboratory, we have found it most effective to combine offline degassing and sonication. The additive effects of the two approaches are both visibly obvious and unforgettable. As a demonstration, I suggest preparing a 50:50 mixture of acetonitrile and water, and placing the mixture under vacuum until no bubbles are observed. Then, while the bottle is still under vacuum, place the bottle in an ultrasonic bath. A vigorous rush of bubbles will be observed, and then completed within seconds. Although we only use inline degassing (see below) for routine degassing of solvents on LC instruments in my lab, the combination of offline vacuum degassing and sonication is still very useful in situations where a complementary degassing approach is needed. For example, if one suspects that the inline degasser in a pump is not working, and leading to bubble formation, degassing the solvent offline can enable a very informative test of the rest of the system to rule out the possibility that the inline degasser is the source of the problem.

A simple block diagram illustrating the use of inline vacuum degassing in LC pumps is shown in Figure 2. In this approach, the solvent is drawn from the bottle through a polymeric tube situated inside of a vacuum chamber on its way to the inlet check valve of the pump (in the case of a high pressure mixing design) or a proportioning valve (in the case of a low-pressure mixing design). The polymer chosen for the tubing inside the vacuum chamber is one that is permeable to the major gases dissolved in the solvent that we need to remove (mainly nitrogen and oxygen), such that gas molecules are drawn across the tube and removed from the liquid selectively. Following the discovery in the early 2000s of particular polymers with enhanced permeability for these gases (7), inline vacuum degassing has become the dominant means of degassing used in LC instruments today. In general, it is easy to use (no manipulation by the analyst) and quite robust (except for an occasional vacuum pump failure or leaky fitting). One important aspect to keep in mind when using inline degassers is that molecules other than dissolved gases that are small and volatile can also cross the tubing wall and be swept away by the vacuum pump. When using pre-mixed solvents (that is, mixed in the solvent bottle so that a mixture such as methanol/water enters the degasser), this means that the composition of the solvent exiting the degasser module will be different from the composition of the solvent entering the module, due to preferential loss of the more volatile component of the solvent. For example, an acetonitrile/water mixture will contain a little less acetonitrile exiting the degasser than it did entering it. For most applications these changes will not be large enough to affect method performance, but some applications that are more sensitive to solvent composition may be affected. Similar effects on the solvent composition occur with other degassing methods, including sparging and offline degassing, so this is not a problem unique to inline degassing.

Older models of inline vacuum degassers relied on long lengths of less permeable tubing compared to the polymers used in newer models. A practical consequence of this is that the volumes of liquid inside older degasser modules (sometimes referred to as the holdup volume of the degasser) were much larger than they are now. This can be important for two reasons: 1) when working with a newer instrument, the analyst may not need to flush the degasser as long as they had to with an older module when changing solvents; and 2) conversely, when moving to work with an older instrument or an instrument from a different vendor, the degasser flushout time used when changing solvents may need to be adjusted to account for different holdup times. In every case, users should either consult the user manual for the particular model of degasser they are using to find out what the degasser holdup volume is, or a find a recommendation for the flushout volume for the degasser when changing solvents.

In addition to the effect of dissolved gases on bubble formation and pump performance, these gases can also affect detection in LC. For example, Brown and coworkers showed that there can be a 400 mAU difference in the absorbance of methanol at 210 nm when the solvent is fully degassed compared to when it is saturated with air (6). Thus, if degassing efficiency varies over time, this variation can lead to detector baseline drift (long-term changes) or waves in the baseline (short-term changes). Moreover, Bakalyar and coworkers showed that changes in the level of dissolved oxygen in the mobile phase significantly affected the fluorescence signal for some analytes in an application focused on the quantitation of polyaromatic hydrocarbons (4). Here, variations in degassing efficiency could have significant effects on the quantitative performance of methods using fluorescence detection.

In this installment of “LC Troubleshooting,” I’ve discussed the importance of degassing solvents used for liquid chromatography, as well as older and newer approaches to the task. The primary problem that can be avoided with proper solvent degassing is bubble formation, which can cause erratic or even no flow from the LC pump. While inline vacuum degassing is currently the dominant approach used in commercially available instruments, there certainly are situations where older approaches, including sonication and offline degassing, are still useful. Understanding the history of these practices, and how they have evolved over time, is a useful facet of knowledge for aspiring LC troubleshooters.

(1) Dolan, J. W. Mobile Phase Degassing: What, Why, and How. 

(2) Dolan, J. W. How Does It Work? Part II: Mixing and Degassing. 

Troubleshooting LC Systems: A Comprehensive Approach to Troubleshooting LC Equipment and Separations

(4) Bakalyar, S. R.; Bradley, M. P. T.; Honganen, R. The Role of Dissolved Gases in High-Performance Liquid Chromatography. 

(5) Stoll, D. R. Mixing and Mixers in Liquid Chromatography – Why, When, and How Much? Part I, The Pump. 

(6) Brown, J. N.; Hewins, M.; Van Der Linden, J. H. M.; Lynch, R. J. Solvent Degassing and Other Factors Affecting Liquid Chromatographic Detector Stability. 

(7) Sims, C.; Gerner, Y.; Hamberg, K. Vacuum Degassing. US6248157B1, June 19, 2001.

Events

Column: LC Troubleshooting

Mobile phase degassing has become very useful for troubleshooting problems with pumps and detection.

The Evolution of LC Troubleshooting: Degassing

This installment of “Sample Prep Perspectives” reports the results obtained from a new survey on sample preparation techniques. This is our first survey since 2016 and its findings are compared with the results of previous surveys dating from 1991. Results involve undercover trends in the field over the last generation and their impact. The survey investigated trends in areas such as which technologies are currently being used, sample loads, sample sizes, automation, the use of solid-phase extraction (SPE) devices (cartridges, disks, plates, tips), SPE chemistries, selection criteria, and problems encountered. Respondents were also asked about sample preparation technologies on the horizon.

periodically surveys readers to uncover trends in the techniques, practice, and applications of sample preparation for chromatography. This year’s results are the seventh in this series (1–6). During these 32 years, several new technologies, including stir-bar sorptive extraction (SBSE), QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction, pressurized liquid extraction (PLE), and others have been introduced. Sample preparation needed to keep pace with developments in chromatography, including the more widespread use of mass spectrometry (MS) with chromatography, improvements in chromatography columns, such as exhibited in the increasing application of ultrahigh-pressure liquid chromatography (UHPLC), and the continued push for more highly sensitive methods (for example, the U.S. Environmental Protection Agency [EPA]-proposed method to monitor perfluoroalkyl substances [PFAS] in drinking water at levels of four parts per trillion). Researchers promoting sample preparation in 1991 are approaching retirement or have already retired, while the new generation of laboratory workers has hopefully gained an appreciation for sample preparation, which was missing a generation ago. Thus, this year’s survey is a golden opportunity to look at how the field has changed with regard to the increased attention to sample preparation over the last 30 to 40 years.

Similar to previous iterations of the survey, the publishers of 

distributed an internet-based survey to a statistically representative group of readers in March 2023, using a similar set of questions to the prior surveys. The response increased compared to previous surveys (541 respondents). During this month’s installment of Sample Preparation Perspectives, we will look at general trends that have emerged and the current state of the field. In the January 2024 column, we will present Part II of the survey, focusing on trends in automation and technique-specific survey results.

The survey audience was largely similar to previous years, though there are some differences in their fields of work. Far fewer respondents came from the pharmaceutical and over-the-counter drug sector than 10 years ago (6.4% in 2023 compared with 25.5% in 2013), while more worked in the environmental (15.5% vs. 13.8%), organic chemicals (15.0% vs. 6.1%), and medical/biological (11.3% vs. 9.1%) sectors. Figure 1 summarizes the self-reported fields of survey respondents in the 2013 and 2023 surveys. There is a good distribution of workplace organizations from which survey participants come, as depicted in the Pareto chart in Figure 2. Compared to 2013, only about a third as many respondents come from private industry (16.4% in 2023 compared to 47.7% in 2013), but that amount is made up by increased numbers of respondents employed by research institutes and independent/contract laboratories. While the plurality of survey respondents still come from research and development positions, there are fewer research and development scientists and laboratory managers among the survey respondents than a decade ago, though those in academia, technical support, quality control, regulatory, engineering design, and process control all increased. These results, shown in Figure 3, demonstrate a nice distribution of job functions, with a balance between laboratory workers and supervisors, research and routine analysis, and other functions. Finally, we’ve never previously determined the geographic distribution of those who respond to our survey. This distribution seems to be representative of analysts globally, with 32.8% of respondents coming from North America, 27.5% from Central and South America, 24.5% from Europe, 12.0% from Asia, Australia, and the Pacific Rim, and 3.2% from Africa and the Middle East.

In the original 1991 survey, Majors provided a baseline for the status of the field of sample preparation, establishing the rationale for the research which advanced the area (1). Notably, he queried surveyed researchers on their opinion of the importance of sample preparation. (For purposes of these, and most other, discussions, sample preparation is viewed as the combination of all steps between the sample collection and storage steps and the actual analysis [that is, chromatography and detection]). Nearly all respondents (91%) felt sample preparation was a very (60%) or moderately (31%) important step in the analytical process. This reiterated the axiom borrowed from computer programming: “garbage in, garbage out.” This question was not asked in subsequent surveys, so after the development and adoption of new sample preparation technologies, automation, and other advances, does this strong opinion still predominate? After 32 years, chemists still have an overwhelmingly (84.5%) positive opinion of the importance of sample preparation. However, as observed with Figure 4, this opinion has moderated to some extent, with the moderately or minimally important categories seeing increased response. Several potential reasons could lead to this. As modern sample preparation addresses concerns such as cost, efficiency, time, and solvent use, the role and importance of these steps becomes less apparent.

Another observation not updated since 1991 was the distribution of time spent in a typical analysis. In 1991, analysts spent ten times as long processing their samples as the actual analysis (61% vs. 6%) and sample collection accounted for a similar amount of time (6%) as the analysis. Data management took the other significant block of time (27%). In the current survey, time is more evenly distributed across these areas. Significant improvements in sample preparation time via automation and newer sample preparation technologies appear to have lowered this time to be on par with the analysis. What is puzzling is the increase in relative time spent on sample collection, which can perhaps be attributed to a decrease in sample processing and analysis times and an increase in the time spent collecting more difficult or complex samples. Another possibility could be that more analysts are involved with the sample collection, as opposed to receiving samples brought to the laboratory by others, demonstrating better knowledge of sample collection requirements. These survey results are represented pictorially in Figure 5.

A new question in this year’s survey asked what influences the purchase of sample preparation products. Looking solely at “very important” factors, products leading to high recovery have the strongest influence (33.0% very important), but, overall, price (31.7% very important, 46.4% important) and time savings (29.2% very important, 47.7% important) are the greatest contributing factors, as displayed in Figure 6. Regarding price, for capital-intensive instrumental systems, it is hoped that those recommending purchase can delineate between the acquisition cost and the amortized cost per sample. Product sterility is less of a concern, likely important mainly for analysts working with biological samples.

Adding a green perspective to chemical analysis has recently become increasingly important, not just an emerging interest. Our survey uncovered an overwhelming majority (88.6%) of respondents concerned with the environmental, health, and safety effects of the solvents used during sample preparation. To address these concerns, 39.0% of respondents are using solventless sample preparation methods like solid-phase microextraction (SPME) or thermal desorption, and 30.8% are using smaller amounts of traditional aqueous or organic solvents. Others (20.6%) are considering direct sample analysis without sample preparation. Only 9.6% of analysts are using nontraditional solvents, like supercritical fluids or ionic liquids. Green solvent selection has been extensively studied and will be the focus of an upcoming Sample Preparation Perspectives column in 2024. Until then, analysts with concerns over the environmental, health, and safety considerations of solvent use may wish to consult resources provided by the American Chemical Society (ACS) Green Chemistry Institute (7,8) or other published solvent selection guides.

Analytical Errors and Problems with Sample Preparation

The 1991 survey also identified the major sources of error in an analytical scheme as sample processing (22.6%), operator (17.1%), and contamination (14.6%). These three items add up to over half (54.3%) of the error in an analytical process and can largely be attributed to the sample preparation steps. That is, in addition to the sample processing, sample preparation is the most labor-intensive step in an analysis, and the solvents, derivatizing agents, and other chemicals used in sample preparation provide opportunity for inadvertent addition of contaminants to the sample. Three decades later these three sources of error still contribute significantly. (Note that in the 2023 survey, participants were allowed to choose two responses to the question “During a typical analysis, what are the major sources of error?”) However, calibration was identified as the largest contributing factor to analytical error in the most recent survey, with integration also one of the major concerns.

These results are shown in Table I. If each survey respondent selected two items for this question, the error contributed by “sample processing” decreased by about half over the last generation. Presumably, we can account for this due to the increased attention being placed on sample preparation in contemporary practice, the development and use of more efficient sample preparation techniques, and the increased use of automation (to be discussed in Part II of this survey). We can also assume that operator error will be minimized with increased use of automation. Perhaps most alarming is the contribution of contamination to analytical error, given the solvent intensity of many sample preparation schemes and the potential for these solvents to introduce contaminants.

Turning to sample preparation, historically time and labor intensity, cost, and lack of reproducibility have been identified as the most frequent sample preparation problems. These were followed by insufficient sample knowledge, poor sample recovery, and contamination. The results from 10 years ago are presented in Table II along with those from the current survey. This year, poor sample recovery, lack of reproducibility, and interpretation of results rose to the top of the frequent sample preparation problems list, followed by cost and a cluster of items, which includes time and labor intensity and insufficient sample knowledge. Looking into these results is both encouraging and perplexing. The significant decrease in time and labor intensity (from 60.8% in 2013 to 23.8% in 2023) is promising and may reflect the advances in the use of modern sample preparation techniques during this time. The numbers of respondents identifying cost (29.3% in 2013 vs. 26.8% in 2023), insufficient sample knowledge (22.7% vs. 22.5%), and contamination (12.4% vs. 13.8%) are largely unchanged. Perplexing is the sharp increase in responses to poor sample recovery (27.6% vs. 37.6%) and lack of reproducibility (24.0% vs. 30.9%). Perhaps a trend toward smaller sample sizes or more difficult sample matrices may explain this observation. Sample size will be discussed in Part II of these survey results, and a discussion of sample matrices will follow.

During each survey, we query the types of sample matrices analyzed on a regular basis. This year’s results, compared with those from a decade ago, are listed in Figure 7. Significant changes occurred over the past decade. Several matrices decreased in the frequency of which they are analyzed, including pharmaceuticals and over-the-counter drugs (36.0% in 2013 vs. 23.3% in 2023), drinking water (18.4% vs. 14.9%), animal and biological tissues (20.9% vs. 13.1%), physiological fluids (18.4% vs. 11.3%), wastewater (17.6% vs. 10.2%), solvents (16.2% vs. 10.9%), and organic chemicals (23.5% vs. 15.3%). Matrices analyzed more frequently include air (8.9% in 2013 vs. 21.5% in 2023), polymers and monomers (16.7% vs. 31.7%), plants and biomass (14.1% vs. 31.3%), inorganic chemicals (8.2% vs. 24.0%), metals (7.8% vs. 19.3%), soils (11.8% vs. 18.4%), foods and food products (11.5% vs. 15.3%), grains (7.1% vs. 12.2%), cosmetics (6.6% vs. 9.3%), and gases (4.7% vs. 9.3%). “Other” sample matrices include biodiesel, illicit drugs, explosives, flavors and fragrances, fermentation broths, and honey, eggs, and dairy. It is probably fair to say that there is a more even distribution of analysts working across these sample matrices.

Figure 8 shows the sample types encountered in the respondent’s laboratories. Most significantly for solids, but in every case, the frequency for each sample type has decreased, indicating that laboratories appear to be more focused on the type of samples which they analyze. The number of sample types per laboratory seems to have decreased from about two in 2013 to one and a half currently. The number of samples analyzed per week per analytical instrument has increased over the last decade, as demonstrated in Figure 9. However, the trend for the next two years is unclear, as 45.4% of survey respondents expect this number to increase and 39.1% expect a decreased sample load.

This observation of fewer sample types, but increased sample loads, is perhaps a result of consolidation of laboratory operations concurrent with the pandemic or a similar systemic change.

The increased emphasis placed on sample preparation over the past generation has led to major changes in the practice of analytical chemistry in the laboratory. The current state of the field was presented, along with errors associated with sample preparation and general sample information. Part II of the survey results will be presented in the January 2024 Sample Preparation Perspectives column and will cover sample sizes analyzed, laboratory techniques used, automation, and solid-phase extraction phases utilized.

(1) Majors, R. E. Trends in Sample Preparation 

(2) Majors, R. E. Trends in Sample Preparation 

(3) Majors, R. E. Trends in Sample Preparation 

(4) Majors, R. E. Trends in Sample Preparation. 

(5) Majors, R. E. Trends in Sample Preparation. 

(6) Raynie, D. E. Trends in Sample Preparation. 

https://learning.acsgcipr.org/guides-and-metrics/solvent-selection-guides/

Column: Sample Prep Perspectives

In this article, the author reports the results from a survey of trends in sample preparation techniques.

Trends in Sample Preparation, Part I: Current State of the Field

Good Laboratory Practice (GLP) is a regulatory requirement to help ensure the quality, reliability, and integrity of pharmacological and toxicological studies during drug development. This article overviews GLP regulations and related public standards, focusing on practices relevant to analytical and bioanalytical chemists.

Nonclinical and clinical studies are the two pillars of new drug development (1,2). Many of these studies are regulated by health authorities, as described in our previous overview article on pharmaceutical regulations (3). Here, we discuss Good Laboratory Practice (GLP) regulation described in Title 21 Code Federal Regulations (CFR) Part 58 (4) that was enacted in 1978 to ensure data accuracy and integrity of nonclinical laboratory studies (5–8). Two types of mandatory nonclinical studies conducted in the preclinical stage on new molecular entities (NMEs) are pharmacology/drug disposition and toxicology (safety) evaluations. Pharmacology and drug disposition studies investigate the pharmacological effects and the mechanisms of action of the drug in animals, providing information on the absorption, distribution, metabolism, and excretion (ADME) of the investigational product (9,10). Toxicology studies monitor the toxicological effects of the drug in vitro (cell cultures) and in vivo (animals) (11).

Although GLP regulations are requirements for conducting pivotal nonclinical safety studies before first-in-human clinical trials, the principles and practices of GLP are often adopted and combined with Good Clinical Practice (GCP) (12,13) for clinical bioanalytical testing (such as those tests for blood, plasma, urine, or tissues collected from test subjects during clinical trials). These public standards are known as Good Clinical Laboratory Practice (GCLP) (14,15).

This paper highlights the principles and practices of GLP relevant to both analytical and bioanalytical chemists supporting nonclinical studies. It is the fifth in nine white papers on “Pharmaceutical Industry for the Analytical Chemist.”

Table I lists the terms and acronyms used in the paper.

GLP in Drug Development and Registration of Other Products

GLP is a quality system covering the required organizational processes and the conditions under which pivotal nonclinical studies are planned, performed, monitored, recorded, reported, and retained (4,5). GLP studies are required to register new chemicals, food, color additives, human and animal drugs, medical devices, biological products, biocides, or agrochemicals (4,5). This paper focuses on drugs for human use. However, agencies such as the United States Environmental Protection Agency (EPA) also issue GLP regulations for products under their purview (16), as promulgated in 40 CFR 160 (FIFRA) and 40 CFR 792 (TSCA).

Regional authorities like the U.S. Federal Drug Administration (FDA), European Medicines Agency (EMA), and Organization for Economic Cooperation and Development (OECD) establish GLP regulations for human-use drugs. In the United States, the FDA has published the GLP in the Code of Federal Regulations (CFR) Title 21 CFR Part 58 (4). Currently, OECD has 38 member countries that have adopted the OECD GLP principles and guidance documents (8). There are EU directives relating to adopting GLP principles, some of which reference OECD-published guidelines (17,18). Although there are many similarities between regional GLP regulations, not all aspects are the same, especially for terminologies (19). As described in our previous paper (3), it is incumbent on the sponsor to fully understand the regulations within the jurisdiction they wish to conduct studies.

We briefly discuss pharmacokinetic (PK) and toxicokinetic (TK) studies, which are nonclinical studies that aim to demonstrate that the drug candidate is safe for initial human testing and exhibits the desired pharmacological activities. Before the drug can be tested in humans, GLP preclinical PK studies should be performed on rodent and non-rodent species. PK studies determine the concentration of the drug candidate within various parts (organs) of the animal at defined time points after drug administration. PK studies aim to obtain bioavailability data (how the drug is absorbed into the systemic circulation) and to allow extrapolation to human equivalent doses (9–11). TK studies also determine the concentration of the drug within different parts of the animal at defined time points after administering the drug. They are designed to aid in the interpretation of toxicity findings. TK studies use exaggerated concentrations of the drug candidate to study how toxic the drug may be (maximum tolerable dose). Preclinical toxicology studies often comprise of acute (single dose), subacute (repeat dose), genotoxicity, and safety pharmacology studies. A 28-day repeat dose toxicology study in rodents and non-rodents is typically needed before an investigational new drug (IND) application for human clinical trials is filed (11,20).

In addition to preclinical PK and TK studies for safety evaluation that should be conducted under GLP, other studies may include general toxicity studies (acute, sub-chronic, and chronic), local tolerance studies, genotoxicity studies, carcinogenicity studies, developmental and reproductive toxicity studies, immunotoxicity studies, analytical studies of biological sample collected from animals, and analytical studies of the dose formulations (21).

Table II lists the ten principles of GLP according to the OECD, with brief descriptions of each (4,8). The reader is referred to the official guidance documents and the company’s internal SOPs, if available.

Although GLP and cGMP regulations have similar goals to ensure data accuracy and integrity and are enforced by the FDA in the United States, there are significant differences (3–5). GLP applies to the preclinical or nonclinical safety testing phase of drug development and focuses on shorter-term projects and standalone studies often conducted in contract research organizations (CROs) (22). The principles of GLP are adopted and followed in bioanalytical testing in clinical studies. cGMP regulations apply to the late-stage drug manufacturing process of drug substances and products, including clinical trial materials and commercial manufacturing (23). cGMP will be further described in our next installment.

Analytical and Bioanalytical Chemists Supporting GLP Studies

Table III lists a nonclinical study’s GLP elements divided into five broad categories: Resources; Practices; Analytical and Bioanalytical Testing; Protocol & Study; and Reporting of Study Events. We provide examples of practice and testing elements relevant to chemists in this section.

Good documentation practice (GDocP) is a core element of GLP compliance (24). Pharmaceutical companies establish their own GDocP aligned with industry standards. For example, the acronym “ALCOA+” is often used to describe components of GDocP: Attributable, Legible, Contemporaneous, Original, and Accurate, along with Complete, Consistent, Enduring, and Available. For the chemist, this means using an electronic laboratory notebook (ELN) to contemporaneously record reagents, solvents, samples, balances, weights, pipettes, and equipment used to prepare and analyze the test and control articles. The analyst must document the technical procedures and data analysis techniques, including the CDS processing method and integration parameters. All raw data and corresponding GLP documentation must be retained and archived as per SOPs.

Chain of custody is essential to maintaining the integrity of the GLP study, so there must be documentation of the delivery, receipt, storage, preparation, analysis, and disposition of the test and control articles. The study director or test facility manager typically enforces these regulatory aspects. The quality assurance unit (QAU) may conduct an in-study inspection of the analytical chemist preparing test and control article samples to assure the correct procedures are followed.

We highlight two specific roles for the analytical chemist working in the laboratory—the analytical chemist responsible for characterizing the GLP test article and monitoring its stability (21 CFR Part 58.105[a] and [b]), and the bioanalytical chemist responsible for analysis of blood, plasma, serum, other bodily fluids, or tissues after administration of the test article to the test systems (21 CFR Part 58.120 [4,22], Part 58.130[a] and [c]). Both roles are essential in supporting GLP studies.

Analytical chemists support GLP studies by developing methods to characterize test articles (drug substances and earlyphase drug product formulations) to determine the identity, strength, purity, composition, or other characteristics that appropriately define it (2,4). Test articles in GLP studies should be characterized and documented using GLP-compliant methods and procedures (21). The chemist uses phase-appropriate method validation for GLP studies (3,21) because there is often little data available on degradation products and pathways at this early stage of development. In addition, a toxicology study is often conducted using simple formulations or carriers before a final drug product formulation is fully established. High performance liquid chromatography (HPLC) with UV detection is commonly used for testing small molecule test articles. The equipment must have proper qualification documentation for a GLP study (7).

Stability of the test and control articles (such as the drug substance and dosing solution when applicable) must be established to assign appropriate storage conditions and assure that the concentration of the test article does not change outside of the established ranges from the date of manufacture through the end of the study.

Bioanalytical methods employed in GLP studies are validated in accordance with International Council for Harmonization (ICH) M10 (25). The guidance applies to bioanalytical methods used to measure concentrations of chemical and biological drug(s) and their metabolite(s) obtained in nonclinical TK studies, nonclinical PK studies conducted as surrogates for clinical studies, and all phases of clinical samples.

Bioanalytical chemists may use techniques such as LC–mass spectrometry (LC–MS), enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence, or surface plasmon resonance to measure the concentration of drug and metabolite in a particular biological matrix such as whole blood, serum, or plasma (26,27).

Core bioanalytical assay validation typically consists of evaluations of the method’s precision, accuracy, selectivity, and specificity. Additional validation experiments are performed to characterize the assay and evaluate the stability of the analyte in the appropriate biological matrix. For example, selectivity must be evaluated in relevant patient populations, presenting a challenge for projects with rare disease indications (26). Stability in the biological matrix is determined over an appropriate concentration range for the assay being validated. Typically, stability is evaluated at room temperature, frozen, and after multiple freeze-and-thaw cycles (25). It is also sometimes necessary to evaluate the assay’s performance in the presence of co-administered medications or elevated levels of the endogenous target (26).

GLP bioanalytical support of large molecule clinical studies also includes immunogenicity testing (28,29). Immunogenicity is the tendency of a drug candidate to elicit an immune response to itself, resulting in the possibility of altered PK or potential adverse clinical events. Immunogenicity testing typically consists of screening, confirmatory, titration, and neutralization assays to identify and characterize the immune response fully (30).

Analytical chemists are essential in determining the concentration, homogeneity, and stability of test articles and reference articles for nonclinical studies. Bioanalytical chemists are essential in analyzing specimens from nonclinical test systems and clinical test subjects. Compliance with GLP ensures the reliability and integrity of these studies and the accuracy of the data submitted to the regulatory authorities.

This paper provides a high-level overview of GLP regulations and related standards for pharmaceutical scientists in nonclinical and clinical studies. Although the goal of the regulation is straightforward, compliance activities are diversified and variable, driven by the company’s standard operating procedures (SOPs), resources, and compliance culture. The information presented here stems from health regulations, guidance documents, and the authors’ experience. Presenting a concise overview of this complex topic is challenging. The reader is referred to cited references and the company’s internal SOPs for more definitive compliance activities in the laboratory.

The authors thank the reviewers for providing timely technical and editorial inputs to this article: Kirk Smith of Smith & Co. Consulting; Alice Krumenaker of Hovione; Mike Shifflet from Kenvue; Doug McDowell of Eurofins Biopharm Products Testing; Kate Evans of Longboard Scientific Consulting; Bob McDowell of McDowell Consulting; David VanMeter of Sartorius; Michael Swartz of Karuna Therapeutics; Chris Tudan of Veloxity Labs; Aimin Tan of Nucro-Technics; and He Meng of Sanofi.

Drug Discovery and Development: Technology in Transition, 

(2) Dong, M. W. Drug Development Process: Nonclinical Development of Small Molecule Drugs, 

(3) Doneski, L., Dong, M. W. Pharmaceutical Regulations: An Overview for the Analytical Chemist, 

(4) Code of Federal Regulations, Title 21, Part 58, 

Good Laboratory Practice for Nonclinical Laboratory Studies

https://www.ecfr.gov/current/title-21/chapter-I/subchapter-A/part-58

(5) Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Market Authorization for Pharmaceuticals. US FDA, 2019.

Good Clinical Practice: Pharmaceutical, Biologics, and Medical Device Regulations and Guidance Documents Concise Reference, 

Good Laboratory Practice for Nonclinical Studies (Drugs and the Pharmaceutical Sciences)

(8) OECD Principles of Good Laboratory Practice, Environment Directorate Organization for Economic Cooperation and Development, 1998, DOI: 

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Winter’s Basic Clinical Pharmacokinetics, 

6th ed. Lippincott Williams & Wilkens, 2017.

 Casarett & Doull’s Essentials of Toxicology, 

(12) Kolman, J.; Meng, P.; Scott, G. (Ed.), 

Good Clinical Practice: Standard Operating Procedures for Clinical Researchers

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(14) Ezzelle, J; Rodriguez-Chavez, I. R.; Darden, J. M.; D’Souzac, M. P., et al. Guidelines on Good Clinical Laboratory Practice (GCLP): Bridging Operations between Research and Clinical Research Laboratories, 

(15) Good Clinical Laboratory Practice, World Health Organization. DOI: 

(16) Code of Federal Regulations, Title 40, Part 160, 

https://www.ecfr.gov/current/title-40/chapter-I/subchapter-E/part-160

(17) Directive 2004/9/EC of the European Parliament and the Council of 11 February 2004 on the Inspection and Verification of Good Laboratory Practice (GLP).

(18) Directive 2004/10/EC of the European Parliament and of the Council of 11 February 2004 on the Harmonization of Laws, Regulations, and Administrative Provisions Relating to the Application of the Principles of Good Laboratory Practice and the Verification of Their Applications for Tests on Chemical Substances.

OECD and USA GLP Applications, Ann Ist Super Sanita. 

(20) Chen, Z. Good Laboratory Practice (GLP) 101–Regulations and Basic Studies, CDER Inspections of Good Laboratory Practice, Animal Rule, and Bioavailability/Bioequivalence Study Sites – July 19, 2022

Principles of Good Laboratory Practice and Nonclinical Development in Fundamentals of Pharmaceutical and Biologics Regulations: A Global Perspective.

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(22) Andola, H; Purohit, V. K., Good Laboratory Practice Requirements: An Overview. 

(23) Code of Federal Regulations, Title 21, Parts 210 and 211, 

Current Good Manufacturing Practice for Finished Pharmaceuticals,

https://www.ecfr.gov/current/title-21/chapter-I/subchapter-C/part-211

(24) TRS 966 - Annex 5: WHO Good Data and Record Management Practices. Annex 5, WHO Technical Report Series 966, World Health Organization, 2016

(25) ICH M10, Bioanalytical Method Validation and Study Sample Analysis. ICH, 2022.

(26) Stevenson, L., Kelley, M., Gorovits, B., et al.. Large Molecule Specific Assay Operation: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Team, 

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(28) Williams, K; Erickson, R; Fischer, S. K.; Overcoming Disease-Specific Matrix Effect in a Clinical Pharmacokinetic Assay Using Microfluidic Immunoassay Technology. 

(29) Law, W. S.; Genin, J. C.; Miess, C. et al.. Use of Generic LC-MS/MS Assays to Characterize Atypical PK Profile of a Biotherapeutic Monoclonal Antibody, 

(30) Immunogenicity Testing of Therapeutic Protein Products — Developing and Validating Assays for Anti-Drug Antibody Detection. US FDA, 2019.

Perspectives in Modern HPLC

GLP helps ensure the quality of different studies during drug development. Here, the authors outline practices relevant to analytical and bioanalytical chemists.

Good Laboratory Practice (GLP): An Overview for the Analytical Chemist

Complex multispecific antibodies overcome the limitations of standard monoclonal antibodies (mAbs), and have complex three-dimensional structures, greater heterogeneity, and often novel disulfide linkages and unusual post-translational modifications. As a result, the comprehensive characterization essential to the development of cost-effective manufacturing processes and safe and efficacious cancer treatments is much more challenging using traditional analytical instruments and techniques. Fortunately, there are new solutions available that provide fast results, often with greater sensitivity and resolution, even for these novel, complex molecules. The application of various new methods and instruments for the analysis of multispecifics is discussed as a means of highlighting their ability to facilitate the development of these important new modalities.

Targeted anti-cancer agents and immunotherapies have revolutionized the way in which cancer is treated today, but standard monoclonal antibodies (mAbs) do have limitations in selectivity and efficacy. Scientists are overcoming some of these issues through antibody engineering of whole mAbs or parts of mAbs fused to one another or other protein scaffolds. Incorporation of multiple binding sites or using specific fragments from conventional antibodies affords greater in vivo stability, more effective targeting, and greater efficacy.

Multispecific antibodies have complex three-dimensional structures and greater heterogeneity than traditional mAbs. They often include novel disulfide linkages and unusual post-translational modifications (PTMs). Consequently, the comprehensive characterization essential to the development of cost-effective manufacturing processes and safe and efficacious cancer treatments is much more challenging using traditional analytical instruments and techniques.

Full product quality assessment of multispecifics includes molecule identification, the identification of high-molecular-weight fragments and the percentage of heterodimers, determination of purity, charge heterogeneity and sequence variants, analysis of glycosylation and glycation, confirmation of molecular integrity, including sequence and disulfide linkage determination, and the identification of post-translational modifications, such as oxidation, deamidation, and isomerization.

New solutions are coming to market to provide faster results, greater sensitivity, and higher resolution, even for these novel, complex molecules. Fast glycan analysis using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection facilitates characterization, quantitation, and sequencing of many glycans in peptibodies/fused proteins (1). Parallel processing of multiple samples simultaneously using multi-capillary CE approaches accelerates analysis and dramatically shortens new therapy development timelines, while still providing sensitive, high-resolution data (2). Rapid capillary zone electrophoresis (CZE) enables ultrafast charge variant analysis of next-generation antibodies with high-resolution (3).

Differential mobility separation (DMS) technology coupled with MS analysis eliminates the need for chromatography, thereby increasing the throughput of subunit analyses (4). New trapping and fragmentation technologies, meanwhile, enable MS analyses with increased sampling efficiency for greater fragment ion coverage and better sensitivity (2,5–7).

For glycan analysis of the asparagine-linked oligosaccharides of the Fc-Fusion protein etanercept, carbohydrate release and APTS labeling was accomplished using the Fast Glycan Sample Preparation and Analysis kit (SCIEX) (1). CE-LIF was performed using the PA 800 Plus system (SCIEX), equipped with a solid-state laser-based fluorescent detector (λ

 = 520 nm). A 20 cm effective length (30 cm total length; 50 μm i.d.) bare fused silica capillary was filled with the high resolution separation gel buffer (HR-NCHO) system. Pre-injection of water (3.0 sec/5.0 psi) was followed by 2.0 kV/2.0 sec sample injection and finally the introduction of the bracketing standard (2.0 kV/2.0 sec). The 32 Karat software, version 10.1 (SCIEX) was employed for data acquisition and analysis.

Emicizumab (Genentech, Inc.) was analyzed on the BioPhase 8800 system from SCIEX with cartridges using the BioPhase CE-SDS Protein Analysis and cIEF kits (2). Data were acquired and processed using the BioPhase software, version 1.0.

Samples for CE-sodium dodecyl sulfate (SDS) were prepared following the standard SDS-molecular weight (MW) analysis assay kit protocol and analyzed (following capillary conditioning) at 25 °C using the BioPhase bare fused silica (BFS) capillary cartridge and UV absorbance detection at 220 nm.

For cIEF, emicizumab at a 5 mg/mL concentration was mixed with wide-range (W) and narrow-range (N) ampholytes at ratios of 7:1, 5:1, and 3:1 W/N (v/v) to improve the separation resolution of charge variants. Each sample mixture was mixed thoroughly at room temperature,and 100 μL aliquots were placed in the injecting well positions of the injection sample plate. Analysis was performed (after capillary conditioning) at 20° C using the BioPhase neutral capillary cartridge and ultraviolet (UV) absorbance detection at 280 nm.

Ultrafast Charge-Variant Analysis with CZE

Ultrafast charge variant analysis of emicizumab (Genentech) and blinatumomab (Amgen) was performed on the PA 800 Plus 

system using the EZ-CE Pre-Assembled Capillary Cartridge and the CZE Rapid Charge Variant Analysis Kit (3). All data were acquired and processed by the 32 Karat software, version 10.1.

Emicizumab CZE charge heterogeneity analysis conditions were: 30 cm total capillary length (20 cm effective, 50 μm i.d.); applied electric field strength: 1000 V/cm; separation temperature: 25 °C; and pressure injection at 5 psi for 10 sec. The blinatumomab sample (0.2 mg/mL) was washed three times with 100 μL of water through a 10 kDa cut-off centrifugation filter (Millipore) at 14 000 x g to eliminate matrix-related effects. Separation conditions were the same, with the exception of pressure injection at 5 psi for 20 sec.

Three-day forced degradation of emicizumab (1 mg/mL protein solution in 100 mM Tris-HCl buffer) was performed at pH 8.7 and 45 

MS Using Differential Mobility Separation (DMS) Technology

A sample (10 mg/ml) of a bsAb (Merck Healthcare KGaA) was reduced at room temperature using tris(2-carboxyethyl)phosphine then diluted to a final concentration of 0.1 mg/mL in 10% acetonitrile/1% formic acid (v:v) (4). Chromatography (10 μL/1 μg injection) was performed on a MassPREP desalting column (2.1 mm × 5 mm, 20 μm, 1000 Å) at 80 °C using two mobile phases (A: Water + 0.1% formic acid, B: acetonitrile + 0.1% formic acid) with a flow rate of 500 μL/min. MS was performed using an ExionLC system coupled to a TripleTOF 6600 system equipped with an IonDrive Turbo V source and SelexION DMS device (temperature: 150 °C; separation voltage: 3500 V; compensation voltage: -9 / -3 / 9 V). Data were processed using SCIEX OS software, version 1.6.1 with the BioToolKit and BPV Flex 2.0 software.

MS Using Zeno Trapping and Tunable Electron Fragmentation Technologies 

For peptide mapping of a trispecific antibody (5), the sample was denaturated with 7.2 M guanidine hydrochloride in 100 mM Tris buffer pH 7.2, followed by reduction with 10 mM DL-dithiothreitol and alkylation with 30 mM iodoacetamide. Digestion was performed with trypsin/Lys-C enzyme at 37 °C for 16 h. Chromatography was performed using a CSH C18 column Waters Acquity CSH C18 column (2.1 × 100 mm, 1.7 μm, 130 Å) at 50 

C using an ExionLC system, with 10 μL (4 μg) of the trypsin/Lys-C digest separated. Mobile phases A and B consisted of 0.1% formic acid in water and acetonitrile, respectively. A 60-min gradient profile was used at a flow rate of 300 μL/min. MS data were acquired with a data-dependent acquisition (DDA) method using the ZenoTOF 7600 system with either CID or EAD as fragmentation mode and processed with Byos software (Protein Metrics Inc.). General method parameters were kept the same between the two methods, but the electron energies for the CID and EAD cells were rolling and 7 eV, respectively.

Peptide mapping of etanercept (reference material #8671, NIST) (6) was performed on a sample prepared in the same manner, except that the digest was further treated with an endoglycosidase (PNGase F, Agilent Technologies Inc.) for removal of all N-glycans followed by reaction with SialEXO (Genovis Inc.) to cleave sialic acids. For chromatography, the same column, instrument, and mobile phases were used, but the sample size was 3 μL (4 μg), the column temperature was 60 

C and the flow rate was 250 μL/min. MS data were acquired using a 10 Hz DDA method on the ZenoTOF 7600 system with the electron energy for the EAD cell set to 7 eV. The data were processed with Byos software (Protein Metrics Inc.).

Glycan Characterization, Quantitation, and Sequencing

Accurate glycan analysis is essential because glycan microheterogeneity, charge heterogeneity, and product-related impurities impact the safety, stability, and efficacy of antibody-based therapeutics. Typical LC- and CE-based methods require labor intensive, time-consuming sample preparation including glycan release, purification (multiple centrifugation steps), fluorophore labeling, and pre-concentration prior to analysis.

A new method that uses a high-resolution gel (HR-NCHO) buffer enables excellent separation performance even with a simple preparation protocol that requires just one hour for glycan release, fluorophore labeling, and clean-up and 5 min for CE-LIF analysis of the fluorophore-labeled glycans (8). The protocol leverages a novel magnetic bead- mediated process that affords reproducibly high yields (9,10). Because there is no need for centrifugation or vacuum-centrifugation, the method is suitable for single analyses, and can also be used with automated liquid handling platforms that process up to 96 samples at a time (11). Use of a triple-internal standard approach and specialized software allows structural assignment of the separated glycans (12).

The utility of the method for assessment of N-glycan microheterogeneity was demonstrated for the National Institute of Standards and Technology monoclonal antibody (NIST-mAb) reference material (RM 8671) (13).

Glycan analysis of the asparagine-linked oligosaccharides of the Fc-Fusion protein etanercept (APTS and 2-AB labeled etanercept N-glycans) using the fast glycan method was also compared to analysis using traditional ultrahigh-pressure LC (UHPLC) with fluorescence (FL) detection (1). Etanercept has three N-linked glycosylation sites, one at the Fc domain and two at the TNFα receptor domain of the molecule. The UHPLC analysis took nearly 40 min and identified 12 glycan structures. The CE-LIF analysis revealed 18 glycans with fast separation in <3.5 min. Even better resolution could be achieved for the CE-LIF analysis when a longer capillary (50 cm effective length) was employed.

CE offers high-resolution and robust separation of molecules and is an invaluable technique for the characterization of complex biotherapeutics. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE, also referred to as CE-SDS) can monitor protein size variants caused by artificial fragments resulting from insufficient alkylation (14). With temperature optimization, it is possible to fine tune the separation performance for even complex multispecifics (15). cIEF can monitor protein charge variants caused by PTMs that are considered CQAs due to their influence on protein stability and potency (16). Indeed, cIEF can separate charge variants with very close isoelectric point (pI) values and is applied in quality control (QC) and lot-release tests in the biopharmaceutical industry (17).

The ability to process multiple CE samples simultaneously while still benefitting from the high resolving power and excellent quantitation and reproducibility of CE can accelerate method and process development for multispecifics (2). Parallel processing allows the implementation of a design-of-experiment (DoE) approach for the development of an optimized CE-based method. It also enables more rapid analysis of larger quantities of samples generated during DoE studies pursed as part of process development activities. If the multiplexed CE system enables seamless transfer of optimized methods to single-analysis instruments employed for QA/QC and thus in a GMP environment, scaleup can also be accelerated.

To demonstrate the value of parallel CE processing, comprehensive characterization of emicizumab, a biospecific antibody (bsAb) used for the treatment of hemophilia A, was performed via CE-SDS and cIEF using a multi-capillary CE system (2). With the employed workflow, CE was leveraged to achieve standard characterization of protein size and charge variants using a one-platform system with high reproducibility and throughput.

Emicizumab has an asymmetric structure with heterogeneous heavy chains (HC) and a common light chain (LC). Reduced and nonreduced forms were analyzed for purity using CE-SDS (Figure 1). The heterogenous HCs were not separated, as indicated by a single peak in the electropherogram, and no high-molecular-weight species (HWS) were detected. The electropherogram of the reduced form revealed a low-molecular-weight species (LWS) that could be the non-glycosylated heavy chain. The intact form emicizumab (HHLL) was determined to be greater than 92%, and the LC to HC was approximately 2:5.

Excellent repeatability was observed for both SDS and cIEF analyses of emicizumab across capillaries and injection replicates, expediting the optimization of parameters that can affect separation performance. The pI values of the charge variants of emicizumab lies in the range of 6–7, indicating that a narrow range ampholyte of 6–8 can help improve separation resolution. Various combinations of wide-range ampholytes (3–10) (W) and narrow-range ampholytes (6–8) (N), including W/N of 3/1, 5/1, and 7/1 (v/v) were tested for resolution improvement. Figure 2 shows that N led to improved resolution but increased noise.

As a next step, full factorial DoE with 4 factors (pH and concentration of the anolyte, catholyte, and chemical mobilizer solutions and concentrations of urea and cathodic and anodic stabilizers), each with 2 input levels, would require 16 runs for optimization. On a single-capillary system that would take approximately 18 h to complete, whereas with a multiplexed system with eight capillaries, only two runs would be necessary.

Charge heterogeneity analysis of multispecifics is important during product development, production, stability, and release testing (18). As many as 15 variants can be generated in a single batch of a multispecific antibody, with perhaps two of them being therapeutically relevant. Any method for charge heterogeneity analysis must therefore be high resolution, rapid, accurate, and reproducible. Commonly used ion exchange chromatography and isoelectric focusing techniques tend to be relatively slow, however (19,20).

CZE using nanoliter injection volumes separates analytes based on their charges and hydrodynamic volume-to-charge ratios (3). It can be used for the separation of large and small molecules with a wide range of chemical and physical properties and offers plate numbers approximately several dozen times greater than that of high performance liquid chromatography (HPLC). Analysis times are typically 10 min for next-generation protein therapeutics in their native forms.

When the bsAb emicizumab was diluted with water to a concentration of 1 mg/mL and six consecutive CZE analysis runs were completed in less than 8 min each, six peaks were observed (two faster-migrating, possibly basic species, three slower-migrating, possibly acidic species, and the main peak for the bsAb) with high reproducibility (3).

A low-concentration (0.2 mg/mL) sample of the BiTE bsAb blinatumomab required buffer exchange prior to injection for CZE analysis due to interference of the initial buffer (3). Five peaks were revealed for this bsAb (Figure 3). Since CZE runs typically take <10 min, it is recommended that analysis first be performed without any sample preparation, and if insufficient separation is observed, a simple buffer exchange (washing with water) should eliminate any matrix effects.

A three-day forced degradation study of emicizumab at pH 8.7 and 45 °C was also performed (3). CZE and cIEF analyses were both performed (Figure 4),because cIEF can be used to determine isoelectric point (PI) values (although the run time for cIEF is typically 30–40 min). These results demonstrated that CZE can be readily applied to rapid, high-throughput charge heterogeneity profiling; results were obtained five times faster than those for cIEF with apparently very similar relative peak areas for the main peak.

It should also be noted that, when combined with MS, the identification of deamidated peptides following separation from their non-deamidated counterparts can be achieved, which is not possible with LC (3).

MS-Based Subunit Analysis with Increased Throughput Using Differential Mobility Separation (DMS) Technology 

Subunit analysis by LC–MS enables the analysis of the individual chains of bsAbs in a single method, but the chromatographic separation of protein chains prior to MS analysis is challenging and can complicate data analysis. One solution for overcoming this issue is to use differential mobility separation (DMS) technology coupled with MS analysis. This approach was demonstrated for a bispecific protein sample consisting of three distinct amino acid chains (one light chain, one AG chain, and one GA chain) forming two different binding domains (4).

Chromatographic separation of the chains required a run time greater than 20 min. The use of DMS following installation of the DMS cell, which required just 2 min, enabled unambiguous characterization of each chain in a single injection without chromatography for simpler data collection and analysis and reduced analysis time for increased throughput.

For this bsAb, the three different protein chains were separated by serially scanning three compensating voltages (CoVs) within a single injection (4). The total ion chromatograms for each CoV could be visualized separately and the raw data evaluated, providing clean spectra and giving more confidence in the interpretation of the reconstructed results.

New Zeno Trapping and Tunable Electron Fragmentation Technology Impacts 

In traditional time-of-flight mass spectrometry (TOF-MS) instruments, the duty cycle problem arises because fragment ions arrive at the pulser region with a spread of velocities, and only a small percentage get accelerated into the flight tube. With Zeno trap, the ion fragments are trapped and then ejected in order and inversely proportional to their 

 values so that all of them arrive at the TOF pulser simultaneously. More than 90% are injected into the TOF, leading to improved MS/MS ion sampling for all 

 species (10X or more). The result is greater sensitivity for MS/MS and high-resolution multi-reaction monitoring (MRMHR) acquisition scan modes and greater ion fragment coverage (5).

Meanwhile, unlike traditional electron capture dissociation (ECD) for MS applications, which relies solely on electrons with low kinetic energy (0 EV), controlled electron activated dissociation (EAD) enables tuning of the velocity of the electrons, and thus their kinetic energy (5). Not only is electron transfer dissociation at 0 EV possible, but also “hot” ECD mechanisms at 7 EV for peptide mapping of glycopeptides and disulfide-bonded peptides with side-chain preservation for isomer confirmation, as well as electron impact excitation of ions (EIEIO) at 10 EV for analysis of small-molecule organics. Notably, EAD provides similar quantitative performance and sequence data compared to collision-induced dissociation (CID), but with improved fragment ion coverage (6). The EAD reaction is also robust, reproducible, and quantitative and suitable for use with liquid chromatography.

When Zeno trapping is combined with EAD fragmentation, much greater fragmentation coverage and increased sensitivity are realized at fast scan rates of up to 133 Hz, even for data-dependent and MRMHR acquisitions (5).

Peptide mapping of a trispecific candidate with a main binding site and two single-chain variable fragments (SCFVs) with affinity to secondary and tertiary targets shows the value of the combined use of Zeno trapping and EAD fragmentation (5). Confirmation of leucines and isoleucines, even though they differ by just 14 Da, was achieved (Figure 5), which otherwise would require the use of time-consuming and often inconclusive methods, such as DNA sequencing and Edman Degradation (5). It was also possible to resolve aspartic and isoaspartic acids (the result of deamidation of asparagine), which can impact target binding.

Localization data for labile molecules, such as glycans, can be difficult to obtain using CID fragmentation. With EAD, however, the integrity of labile modifications is maintained, allowing for generation of both localization and identity information. As can be seen in Figure 6, confirmation of the site of glycosylation was possible for the trispecific candidate. Detection of oxonium ions helped to confirm the identity of the glycopeptide (5).

Analysis of disulfide bonds was also simplified. Nonreducing sulfide mapping of multi-specifics generally results in large peptides that require additional fragmentation often not achievable with CID, but for which full sequence coverage is possible using EAD with the hot ECD mechanism (5). In the case of the trispecific candidate, 7,000 DA and 11,000 DA peptides were analyzed with excellent coverage across the entire peptides. In addition, peaks for the individual peptides that make up the larger disulfide-bonded peptides were observed.

EAD was also used to confirm the existence of disulfide and trisulfide linkages as well as free thiols in a bsAb in which two different heavy chains and two identical light chains are linked (6). An EAD platform method was developed that provides signature fragments to differentiate disulfide and trisulfide linkages, enabling a higher level of structural characterization in one injection. Increased detection of fragments (by 5- to 10-fold) enabled free thiol detection at low abundances. The reproducibility of EAD fragmentation also enabled analysis of more precursors than other alternative and low-reproducibility fragmentation techniques.

While both EAD and CID were able to identify all expected disulfide linkages from the bsAb, EAD provided higher MS/MS coverage for long peptides (6).

Multispecific antibodies are complex molecules with potential to serve as highly effective, targeted therapies. Ensuring their structural integrity, purity and safety is essential, but comprehensive characterization can be challenging given their greater structural complexity and heterogeneity. The time constraints often associated with accelerated development timelines for breakthrough address the developability gap by more rapidly identifying candidates with the greatest likelihood of success with respect to efficacy, safety, and manufacturability.

Analytical tools that can provide robust, reliable, in-depth information on critical quality attributes in a high-throughput manner are needed so that more detailed data can be gathered to enable informed decision-making at early development stages. Fast glycan analysis, parallel CE processing, CZE, MS leveraging DMS technology, and the use of Zeno trapping and EAD for MS analyses are all examples of advanced analytical techniques designed to enable rapid and robust analyses of even complex multispecifics.

Comparative Characterization of the Fc Domain N-glycosylation in Monoclonal Antibody and Fusion Protein Therapeutics by CGE-LIF and UPLC-FL.

 SCIEX Separations, Brea, CA; Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Hungary, 2018. 

https://sciex.com/content/dam/SCIEX/pdf/tech-notes/all/CE_vs_UHPLC.pdf

(2) Yang, Z.; Santos, M.; Li, T.; Mollah, S. 

One-Platform Strategy for Comprehensive Characterization of the Bispecific Antibody, Emicizumab, Using the BioPhase 8800 System

https://sciex.com/tech-notes/ce/One-platform-strategy-for-comprehensive-characterization-of-bispecific-antibody-emicizumab-using-multi-capillary-BioPhase-8800-system

CZE: Ultrafast Charge Variant Analysis for Next-Generation Antibody Therapeutics.

https://www.chromatographytoday.com/article/bioanalytical/40/sciex/cze-ultrafast-charge-variant-analysis-for-next-generation-antibody-therapeutics/2948

(4) Pohl, K.; Schanz, J.; Tonillo, J.; Mehl D.; Kellner, R.; McCarthy, S. 

Increased Throughput for the Characterization of Bispecific Antibodies on the Subunit Level Using SelexION Differential Mobility Separation (DMS) Technology. 

SCIEX, Framingham, MA, USA; Merck Healthcare KGaA, Darmstadt, Germany; SCIEX, Villebon sur Yvette, France, 2020. 

https://sciex.com/content/dam/SCIEX/pdf/tech-notes/all/selexion-bispecific-antibody.pdf

Accelerating the Characterization of Next-Generation Biotherapeutics with Mass Spectrometry Leveraging Novel Ion Trapping and Alternative Fragmentation Technologies. 

https://www.chromatographyonline.com/view/acclerating-the-characterization-of-next-generation-biotherapeutics-with-novel-ion-trapping-and-alternative-fragmentation 

Comprehensive Analysis of Next Generation Biologics Using Advanced Data Interpretation Software.

https://www.labroots.com/webinar/biotherapeuric-characterization-biologics-explorer-software2 

Comprehensive Characterization of Etanercept Using the ZenoTOF 7600 System

https://www.labroots.com/webinar/comprehensive-characterization-etanercept-using-zenotof-7600-system 

The comprehensive characterization of multispecific antibodies is essential to the development of safe and efficacious cancer treatments.

October 2023 | LCGC North America

The Evolution of LC Troubleshooting: Degassing

Trends in Sample Preparation, Part I: Current State of the Field

Good Laboratory Practice (GLP): An Overview for the Analytical Chemist

Overcoming Complex Analytical Challenges Posed by Targeted Multispecific Antibody Cancer Therapeutics

Health and Safety Concerns of Hydrogen Use with Gas Chromatography