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, we continue the discussion started earlier this year about how some specific troubleshooting topics have evolved over time, this time focusing on chemical causes of peak tailing, and strategies that we can use to overcome them. Understanding how knowledge and technologies related to peak tailing have evolved is helpful for troubleshooting problems with peak shape, 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 “LC Troubleshooting” installments in the last year. So, in September, 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 doesn’t do any harm, but they provide no benefits and 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 knowledge and technologies related to addressing the problem of peak tailing as it relates to basic compounds (that is, compounds that lose protons to the mobile phase when pH is increased) analyzed under reversed-phase (RP) LC conditions. Today, we have a much better—though not fully complete—understanding of the peak tailing phenomenon than we did 20 years ago, and recently introduced column technologies give us more options to try when tailing peaks are observed. It is useful to have a broad sense for how things have changed, as this can impact choices during the development of new methods or the troubleshooting of existing methods. Readers interested in learning more about peak tailing are referred to previous installments of “LC Troubleshooting,” (1,2), Dolan’s book on the subject (3), and a very recent, masterful review article on the topic of peak tailing of basic compounds under RP conditions by McCalley (4).

A Tailing Peak is Just One of Many Possible Poor Peak Shapes

In the June 2022 installment of “LC Troubleshooting,” I surveyed the many different ways a peak can be poorly shaped, including fronting, split, tailing, and negative peaks (5). Although peak tailing is just one way for things to go wrong, it is a very important one, both because it is relatively common in reversed-phase separations of basic compounds, and because it can be one of the more difficult problems to solve. This difficulty is due, at least in part, to the fact that we don’t yet fully understand the tailing phenomenon (see below for more about the state of our understanding), and when we don’t fully understand the problem, our troubleshooting effort is reduced to a mere trial-and-error approach. As was discussed in the November 2021 installment of “LC Troubleshooting,” also about peak tailing in RP separations, we also differentiate between different subtypes of tailing—exponential tailing and overload tailing. Examples of these peak shapes and a comparison to the ideal Gaussian peak shape is shown in Figure 1 (2).

As is shown qualitatively in Figure 1, and quantified using the measure of plate number (

), tailing peaks observed in a chromatogram that is populated with at least some normal peaks will generally be broader than their symmetrical counterparts, particularly toward the base of the peak. This is not desirable for two major reasons. First, the increased width of the peak limits the ability of the method to separate closely eluting peaks, particularly when they appear to the right of the tailing peak, and especially if the neighboring peak is lower in concentration. In these cases, the peak tail like that shown in Figure 1 can effectively obscure a low concentration neighbor. Second, the slow approach of the detector signal to the baseline on the right side of a tailing peak makes it difficult to decide (both for humans and software) where to place the integration endpoint when the method is used for quantitative purposes. This can lead to problems with accuracy or precision of the method. Indeed, this is a major driver for the guidance in Chapter 621 of the 

that specifies that peak asymmetry should be less than 1.8 (6) (see reference [7] for a detailed discussion on the calculation of peak asymmetry; a perfectly symmetrical peak has an asymmetry of 1.0).

While the chemical causes of peak tailing discussed in this article are very important, it is also important to remember that there can be purely physical causes of tailing peak shapes that have nothing to do with the chemistry. These include poor connections (for example, capillaries connected to valves, columns, heat exchangers, or detectors), mismatches between the sample solvent (that is, the diluent) and the mobile phase composition, and a column that is physically failing due to irregular organization of particles inside the column or fouling of the inlet frit, for example. A fuller discussion of these problems and potential solutions can be found in the September 2021 installment of “LC Troubleshooting” (8).

Troubleshooting Advice for Tailing Peaks circa 2000

In his February 1999 installment of “LC Troubleshooting,” titled “Basic Compounds: Starting on the Right Foot,” John Dolan provided a list of suggestions to consider when developing a method for the separation of basic compounds by RPLC (1). This list, which reflected the state of the art at the time, was intended to maximize the user’s chances of getting a good separation from the start. Much of the article was ultimately dependent on knowledge about the nature of silica-based stationary phases at the time, and the central idea that silanol groups (-Si-OH) at the surface of silica particles can lose protons when the mobile phase pH is increased, leading to a population of negatively charged sites on the silica surface.

The number of these negatively charged sites on the surface, and the pH at which they lose their protons, is affected by several factors, including local surface geometries, the concentration of metal impurities in the bulk silica, and specific processing steps used during manufacturing of the stationary phase. In general, the presence of the negatively charged sites is associated with poor tailing peak shapes for basic compounds. Recognizing this, Dolan made several recommendations, some of which are highly relevant today, and some of which have fallen out of favor because of superior alternatives that have been developed in the intervening years.

At the time, a distinction was made between newer “type B” silica and older “type A” silica, with a key characteristic of type B being a much lower metal impurity content. This lower metal level reduced the number of highly acidic silanol groups that would deprotonate well below pH 7.

Understanding that the equilibrium illustrated in equation 1 is affected by pH, and that negatively charged silanol sites can be largely suppressed by working with mobile phases below pH 3 (even for type A silica), working in the pH range of 2 to 3 is generally helpful (9).

Add triethylamine (TEA) to the mobile phase. 

This is the one I vividly remember learning in chromatography school. If something in your sample is interacting with the stationary phase in a bad way, add something to the mobile phase that looks like the analyte. This is the strategy here—adding TEA to an acidic mobile phase effectively floods any negatively charged surface sites with positively charged triethylammonium ions, thereby blocking adverse interactions between analytes that are basic compounds and those sites.

. When all else fails, and if it is suspected that the silica itself is contributing to tailing peaks, try eliminating the silica altogether. This means moving to some other type of column technology, which, in the year 2000, meant choosing a stationary phase based on an organic polymer (for example, polystyrene-divinylbenzene) or particles based on a different metal oxide, such as alumina, titania, or zirconia.

With the exception of the use of TEA, this guidance is still relevant today. To the best of my knowledge, all new silica-based columns introduced into the marketplace today are based on high-purity type B silica; thus, the distinction between types A and B is perhaps not as salient as it once was. The use of TEA, while important in a number of legacy methods, is declining in popularity due to the widespread use of type B silicas, and the introduction of new column technologies (see below) that have made the use of TEA much less necessary. Finally, in most cases, the use of TEA in the mobile phase is considered completely inappropriate when using a mass spectrometric detector.

Improvements in Knowledge and Understanding since 2000

Readers interested in a thorough review of research related to improving our understanding of peak tailing in the specific case of basic analytes separated by RPLC are referred to the recent (and excellent) review article by McCalley (4). He states that while we have learned a lot through the efforts of many different researchers over the past 20 years, “…elucidation of the fundamental principles involved in determining peak asymmetry of bases has vexed the most distinguished researchers in the subject for many years.” In other words, we cannot yet fully explain all the experimental results that have been observed in studies of this problem. Nevertheless, these studies have yielded insights that are practically relevant.

In my view, the most impactful studies during this time have shed light on the factors that influence the overload tailing behavior shown in Figure 1c. Specifically, McCalley’s observation that such overload behavior is observed on silica-based phases both for cationic and anionic analytes is very important. It suggests that not all overload tailing can be explained by implicating the negatively charged silanol sites in a central role in the tailing. This simply does not make sense when the analyte producing the overloaded peaks is an anion. Some other explanation is required. McCalley has proposed a theory of mutual repulsion wherein the adsorption of a small amount of charged analyte (cation or anion) to the stationary phase surface results in repulsion of similarly charged analyte molecules that enter the zone of previously adsorbed analytes, thus leading to broadening of the zone (and the corresponding peak observed at the detector) (10). An important piece of evidence that supports this theory was the observation of such overload tailing even with stationary phases composed completely of organic polymers, which of course cannot have any negatively charged silanol sites (11).

One of the implications of the mutual repulsion theory, if it is correct, is that overload behavior should be strongly dependent on the ionic strength of the mobile phase. Studies from the same time period showed that this is indeed the case (12), and this has important practical consequences, especially when using mass spectrometric detection. Dilute formic acid in water (for example, 0.1%, which is the de facto preferred aqueous mobile phase solvent for ESI-LC–MS) is a low ionic strength solution compared to dilute phosphoric acid or trifluoroacetic acid in water at the same formal concentration. This is because formic acid is a weaker acid, and thus, it dissociates less and produces less of the formate anion and free hydrogen ion compared to stronger acids. Given the low ionic strength of these solutions, severe overload tailing can be observed even at modest analyte concentrations. The good news is that the addition of a modest amount (for example, 10 mM) of ammonium formate (or acetate) to increase the ionic strength of the mobile phase can improve the overloading behavior substantially.

New Column Technologies that Can Be Used to Address Peak Tailing Introduced since 2000

There have been three major developments in column technologies since 2000 that have expanded the list of possible solutions to improve peak tailing of basic compounds when it is encountered.

Introduction of silica-based phases that are stable above pH 7. 

Beginning in the early 2000s, column manufacturers started introducing stationary phases that could be used above pH 10 for extended periods of time. Prior to this time, manufacturers advised that silica-based phases should generally be used in the pH range of 2 to 8 to avoid dissolution of the particles. The ability to work at pH levels above 10 meant that it was possible to develop a method using a mobile phase pH where basic analytes would be deprotonated and neutral (provided p

< 9), rendering all of the discussion above about interactions with negatively charged silanols irrelevant. Over the past 15 years, manufacturers have continued introducing new phases applicable in this application space.

Introduction of organo-silica hybrid phases that reduce surface silanol concentrations. 

A different approach, which moves in the direction of “eliminate the silica” recommended by Dolan in 1999—but not entirely—has been the development of organo-silica hybrid particles that incorporate carbon into the particle structure in place of some of the silicon (13). One effect of this strategy is that the concentration of surface silanols is significantly reduced compared to a typical fully silica-based particle.

Introduction of stationary phases with embedded charged groups that improve overload behavior. 

A more recent trend has involved the development of stationary phases for RPLC with charged groups deliberately introduced into the stationary phase layer. Several manufacturers now carry at least one phase of this design. Although we do not yet have a thorough understanding of the reasons these phases offer the improvements in peak shape for basic compounds that they do, they are increasingly being used to good effect, and it will be interesting to follow their continued development in the coming years. One example of the improvement in overload behavior that can be realized with such a phase is shown in Figure 2. Remarkably, we see that the mass of analyte that can be injected before half of the limiting (that is, non-overload conditions) plate number is lost increases by about an order of magnitude with the charged-surface hybrid stationary phase.

In this installment of “LC Troubleshooting,” I’ve reviewed the importance of peak tailing and some of the chemical causes that can lead to these peak shapes. Some of the strategies for overcoming these chemical causes of peak tailing recommended many years ago, including using high-purity silica, working at low pH, and avoiding silica altogether, are still relevant today. Other strategies, such as using triethylamine (TEA) as a mobile phase additive are slowly declining in importance as new column technologies provide superior solutions. Some of these new technologies, including high pH stable silica-based phases, organo-silica phases, and phases with deliberate incorporation of charged groups, are changing the way we approach method development for basic compounds. 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. Basic Compounds - Starting on the Right Foot. 

(2) McCalley, D. V.; Stoll, D. R. But My Peaks Are Not Gaussian! Part III: Physicochemical Causes of Peak Tailing. 

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

(4) McCalley, D. V. Understanding and Manag­ing Peak Shape for Basic Solutes in Reversed-Phase High Performance Liquid Chroma­tography. 

(5) Stoll, D. R. Essentials of LC Troubleshoot­ing, III – Those Peaks Don’t Look Right. 

(7) Stoll, D. R. But My Peaks Are Not Gaussian! Part I: Basic Concepts in Peak Shape, and the Effect of Extracolumn Connections. 

(8) Stoll, D. R. But My Peaks Are Not Gaussian! Part II: Physical Causes of Peak Asymmetry. 

(9) Méndez, A.; Bosch, E.; Rosés, M.; Neue, U. D. Comparison of the Acidity of Residual Silanol Groups in Several Liquid Chromatography Columns. 

(10) McCalley, D. V. Overload for Ionized Solutes in Reversed-Phase High Performance Liq­uid Chromatography. 

(11) Buckenmaier, S. M. C.; McCalley, D. V.; Euerby, M. R. Overloading Study of Bases Using Polymeric RP-HPLC Columns as an Aid to Rationalization of Overloading on Sil­ica-ODS Phases. 

(12) McCalley, D. V. Rationalization of Retention and Overloading Behavior of Basic Com­pounds in Reversed-Phase HPLC Using Low Ionic Strength Buffers Suitable for Mass Spectrometric Detection. 

(13) Cheng, Y.-F.; Walter, T.; Lu, Z.; et al. Hybrid Organic-Inorganic Particle Technology: Breaking through Traditional Barriers of HPLC Separations. 

(14) Fallas, M. M.; Buckenmaier, S. M. C.; McCal­ley, D. V. A Comparison of Overload Behav­iour for Some Sub 2μm Totally Porous and Sub 3μm Shell Particle Columns with Ionised Solutes. 

Events

Column: LC Troubleshooting

Troubleshooting continues to evolve constantly, changing how we overcome issues such as chemical causes of peak tailing.

The Evolution of LC Troubleshooting: Strategies for Improving Peak Tailing

Molecular spectroscopy techniques, most commonly ultraviolet-visible (UV-vis), are usually thought of as analyzing liquidphase samples, with techniques such as high-performance liquid chromatography (HPLC). Although not as common as in HPLC, molecular spectroscopy can be used with gas chromatography (GC) as well. In this installment, we examine molecular spectroscopy in combination with GC. The two most common techniques used with GC today are Fourier transform infrared spectrometry (FT-IR) and vacuum ultraviolet spectroscopy (VUV). Both provide structural and quantitative information and can be used in complementary fashion with the more common GC–mass spectrometry (GC–MS) and classical detectors. We will discuss the basics of GC–FT-IR and GC–VUV, when and when not to use them, and how they compare to and complement classical detectors.

n a recent installment, we discussed basic care and feeding common to all detectors for GC (1). We saw that there are myriad detectors, and that detection in capillary gas chromatography faces many challenges not seen in traditional spectroscopy. Analytes are in the vapor phase, generally at low (ppm–ppb) concentration, and are moving rapidly, often 50–100 cm/sec, as they are eluted from the column. A detector must have a rapid response, high sensitivity, and ruggedness. The vapor-phase analytes are often semivolatile; they must be maintained in the vapor phase as they exit the column, so the detector is heated to prevent condensation and deposition of the analyte. Chapters in books by Poole and McNair, Miller and Snow, and CHROMacademy, 

’s online training platform, provide overviews and details about detectors (2–4).

Table I provides a summary of many common detectors, grouped into four categories: physical property, ionization, elemental spectroscopic and molecular spectroscopic. The classical thermal conductivity detector (TCD), a physical property detector, is universal, detecting based on the difference in thermal conductivity between analytes and the background carrier gas. Ionization detectors, including electron ionization mass spectrometry (EI-MS) and flame ionization detection (FID), involve ionization of the analyte by various mechanisms and range from highly selective (electroncapture detection [ECD]) to nearly universal (FID). Spectroscopic detectors can be elemental, such as atomic emission (AED) or molecular. Elemental detectors often use high energy techniques to excite analytes. Like the classical spectroscopic detectors used in HPLC, molecular spectroscopic detectors for GC employ lower energy photons that do not ionize or decompose the analytes, and they behave in a very similar manner to their liquid-phase cousins, with a few exceptions. A detailed discussion and analysis of molecular spectroscopic detectors for GC is provided in a 2018 review by Zavahir, Nolvachai, and Marriott (5).

As in HPLC and classical spectroscopy, spectroscopic detectors obey the wellknown Beer-Lambert law, a form of which is shown in equation 1:

 is the molar absorptivity of the analyte, a physical property dependent on the molecular structure, the wavelength of the incident radiation and the sample matrix; 

 is the pathlength of the cell or flow cell containing the analyte; and 

 is the concentration of the analyte. This presents challenges and opportunities particular to gas chromatography that are not seen in liquid-phase analytical techniques. First, the concentration of analyte in GC is necessarily low, as analytes are in the vapor phase and diluted with non-absorbing carrier gas. Low analyte concentration requires a longer pathlength, which provides the main challenge in using optical spectroscopic techniques with GC. The longer pathlength then results in extracolumn band broadening and some resulting loss of resolution and sensitivity.

The vapor-phase analysis required by GC also provides opportunities not easily seen in liquid- or solid-phase analysis. Detection in the vapor phase ensures clean spectra, with no interference from sample matrix or solvent, assuming that the chromatographic separation is complete. It also provides spectral detail and spectral range not generally available when a solvent is present, as we will especially see with vacuum ultraviolet (VUV) detection.

Spectroscopic detectors can provide a combination of qualitative and quantitative information. Infrared (IR) spectroscopy is a classical method for qualitative analysis; we all remember interpreting infrared spectra in our undergraduate training. IR generates signals through interaction of low-energy electromagnetic radiation that excite rotation and vibration of the chemical bonds in molecules. Ultraviolet (UV) spectroscopy has been implemented relatively recently with GC as VUV, using higher energy wavelengths, which excite high-energy electronic transitions. Traditional UV/visible (UV-vis) spectroscopy has been used with GC, but not widely (6). Interestingly, the two most common spectroscopic techniques used with GC straddle the traditional UV and visible range, with IR at lower energy and VUV at higher energy.

VUV operates in the same manner as traditional UV-vis, except at higher energy wavelengths, typically 120–240 nm, while classical UV-vis detectors used in HPLC typically operate from 200–800 nm. VUV gets its name from a classical need for a vacuum to reduce interferences from air, water, and background; however, today’s benchtop and instrument top detectors use a makeup gas to purge air and water, avoiding the need for a vacuum. A GC– VUV instrument operates like a classical UV-vis spectrometer, coupled to the GC. A diagram is shown in Figure 1 (7).

In Figure 1, we see that effluent from the column exit is mixed with makeup gas and flows through a flow cell that serves the same role as the cuvette in a classical UV spectrophotometer. The other components are very similar to the classical instruments and detectors for HPLC. Incident radiation is provided by a modified deuterium lamp, which provides a broad spectrum of electromagnetic radiation to wavelengths as short as 120 nm. After traversing the flow cell, the light is focused onto a diffraction grating, which separates it by wavelength, then to a charge-coupled device (CCD) detector. This configuration allows rapid generation of high-quality UV spectra, enabling VUV as a rapid and highly selective detector.

Figure 2 shows GC–VUV chromatograms of xylene and naphthol isomers. Note the spectral differences between the isomers, which make them easy to identify using a spectral library search. The left side shows the different spectra for the three xylene isomers and for two naphthol isomers. The right side shows a chromatogram of coeluted xylene isomers. Their distinct spectra make them easy to tell apart. In addition to providing qualitative spectral information, VUV also has promising figures of merit with limits of detection in picograms and 3–4 orders of magnitude linear range. It is also fast, with a response time of about 10 ms, making VUV amenable to fast and multidimensional GC.

Nearly all chemists have used infrared spectroscopy, starting as undergraduates and finding it to be a powerful technique, especially in combination with other instrumental and chemical tests for structure determination. There is a long history of 50 or more years of work with GC coupled to FT-IR, yet due mostly to sensitivity concerns, GC–FT-IR remains a niche technique. Figure 3 shows a block diagram of a typical GC–FT-IR instrument. Like the VUV example, it is essentially a traditional spectrometer coupled to the outlet of the gas chromatographic column, with the same caveats discussed above for all detectors applied. The most common interface between GC and FT-IR is a light pipe, which is essentially a heated flow cell coupled to the end of the capillary column. Other interfaces include matrix isolation, in which the column effluent is cryogenically trapped under vacuum onto a gold-plated collection disc, and the related direct deposition, which places an IR microscope close to the column outlet. Matrix isolation and direct deposition provide greater sensitivity, while light pipe is much simpler.

Complementary to mass spectrometry, FT-IR provides both universal and selective detection for organic analytes. Nearly all organic compounds absorb in the typical scanning range of IR spectrometers used with GC (4000–600 cm

) providing universal detection, while scanning a spectrum provides unequivocal structural information. The combination of unique spectra, especially for structural isomers, and structural library searching, along with retention time data, allows highly selective qualitative analysis with GC–FT-IR.

While not as commonly used as ionization, elemental spectroscopic, or physical property detectors, molecular spectroscopic detectors, most commonly FT-IR and VUV, provide useful and complementary capabilities. They are non-destructive, so they can be used in tandem with other detectors. Both FT-IR and VUV are stars for qualitative analysis, especially for difficult problems in isomer analysis not easily addressed by MS. The longstanding problems with using FT-IR with GC, mostly having less sensitivity than MS, still exist and likely relegate FT-IR to a niche detector; however, there is sufficient sensitivity with relatively simple light pipe interfacing for many applications and FT-IR does not require some of the overhead of MS, such as high vacuum. VUV is a relatively new detector that offers sensitivity between FT-IR and MS, combined with a relatively simple instrument to operate and maintain, and excellent selectivity, especially for structural isomers not easily separated by MS. For problems involving structure determination with gas chromatography, mass spectrometry is not the only option; molecular spectroscopy offers benefits that make it worth considering.

(1) Snow, N. H. Basic Care and Feeding of Your Detector. 

Molecular Spectroscopic Detectors for Gas Chromatography

, 2nd ed.; Poole, C. F., Ed.; Elsevier, 2021; pp 371–397. DOI: 

(3) McNair, H. M.; Miller, J. M.; Snow, N. H. 

, 3rd ed.; John Wiley & Sons, 2019; pp 157–175. DOI: 

(5) Zavahir, J. S.; Nolvachai, Y.; Marriott, P. J. Molecular Spectroscopy – Information Rich Detection for Gas Chromatography. 

(6) Gras, R.; Luong, J.; Shellie, R. A. Direct Measurement of Trace Elemental Mercury in Hydrocarbon Matrices by Gas Chromatography with Ultraviolet Photometric Detection. 

(7) Schug, K. A.; Sawicki, I.; Carlton, D. D. Jr.; et al. Vacuum Ultraviolet Detector for Gas Chromatography. 

Column: GC Connections

Gas chromatography can be used in combination with molecular spectroscopy to provide structural and quantitative information on a variety of sample types.

Molecular Spectroscopy: Underutilized Detection for Gas Chromatography

Good Manufacturing Practice (GMP) is the most significant regulation impacting the pharmaceutical industry and requires substantial investment in resources and time commitments. This paper provides a high-level overview of Current Good Manufacturing Practice (cGMP) regulations and related public quality standards. It discusses GMP compliance requirements and practices, emphasizing those relevant to the analytical chemist in development and quality control.

The original cGMP regulations published in 1978 in Codes of Federal Regulations (

210 and 211 (1,2), contain requirements for methods, facilities, and controls for manufacturing, processing, packaging, and testing drug substances and products. The regulations are designed to ensure that a product has the safety, identity, purity, potency, and clinical efficacy it claims to have. 21 

210 is a brief introductory document that provides a general overview of the minimum requirements of GMP in manufacturing, processing, packaging, or holding of drugs, including status, applicability, and definitions of terms. 21 

211 is a longer document that describes the requirements for finished pharmaceuticals and contains eleven subparts from A to K (A. General provisions; B. Organization and personnel; C. Building and facilities; D. Equipment; E. Control of components and drug product containers and closures; F. Production and process controls; G. Packaging and labeling control; H. Holding and distribution; I. Laboratory controls; J. Records and reports; and K. Returned and salvaged drug products). Since technology is continuously advancing, compliance with the most current standards is expected. Thus, the term “current” in cGMP is used. Table I lists the terms and acronyms used in the paper.

GMP regulations provide guidelines and minimum requirements in manufacturing and development practices and facilities to ensure products are consistently safe, pure, and effective for their intended use (1,2). Although the original regulations (21 

210 and 211) are for small-molecule drug products (1–3), GMP compliance is also required for biologics (4), some foods (5), medical devices for human use (6), dietary supplements (7), and other critical quality processes (8,9). Table II lists the U.S. GMP regulations established, amended, and enforced by the US FDA. Other associated regulations from the EMA, public quality standards, and guidelines from ICH, WHO, ISO, and USP were described in our previous paper #4 (10), and they are discussed further here.

The complex and multifaceted GMP regulations and compliance requirements and practices can be found in the regulatory source documents (1–9) and public guidance (10,11), textbooks (12–15), book chapters (16), journal review articles (17–19), and training resources from university programs (for example, regulatory affairs or pharmaceutical sciences) (20) and professional training courses (21). These resources and the company’s quality systems or SOPs should be referred to for definitive guidance in GMP activities.

This paper aims to provide a high-level overview of the cGMP regulations and their principles, intents, and compliance practices. This article is the sixth in a series of nine white papers on “Pharmaceutical Industry for the Analytical Chemist,” to be published in 

from 2022 to 2024. Paper #4 provides an introductory overview of pharmaceutical regulations, public quality standards, and overall compliance processes (10). Paper #5 describes Good Laboratory Practice (GLP) for early-phase nonclinical safety, pharmacokinetic studies, and bioanalytical assays (22).

GMP Compliance: From Regulations to Internal Quality Processes

Since health authorities (regulatory agencies) only address the minimum GMP requirements in the Code of Federal Regulations without providing details on how to comply, the company must understand the regulations for its product category and devise its own quality systems and processes for compliance. To help the reader in understanding these complex and interactive processes, we categorized the compliance scenario into five sections (10):

Pharmaceutical regulations (law of the land promulgated by legislation),

Health authorities (establish and enforce the regulations),

Public quality standards (supplement the regulations in scientific terms),

Pharmaceutical organizations (establish internal quality systems and procedures),

Regulatory trends and approaches (Quality by Design [QbD], risk-based quality systems, and stage-appropriate GMP compliance in drug development).

Figure 1 is a pictorial portrayal of the five categories extracted from reference 10 and revised for GMP regulations.

GMP Regulations from the European Commission

For companies having manufacturing sites in the European Union, EU GMP regulations are listed in the EudraLex guidelines (23), which have eleven sections with some similarities to the US counterparts (1,2).

World Health Organization (WHO) Guidance for Other Countries

Countries or economic regions that do not have their own GMP regulations often reference the WHO guidelines (24). WHO provides numerous guidance documents spanning development through production (25). Considering the diversity of global regulations, the Pharmaceutical Inspection Co-operation Scheme (PIC/S) was established in 1995 and is a non-binding, informal cooperative arrangement between more than 50 regulatory authorities in the field of GMP for medicinal products for human and veterinary use. PIC/S is the sole organization that exclusively deals with GMP and provides harmonized standards and training for inspectors worldwide (26).

Although PIC/S is not a trade agreement, a variety of mutual recognition agreements (MRA) exist between major regulatory agencies such as the FDA, EMA, Health Canada, PMDA, and others. The goals of MRAs include:

Relying on each other’s GMP inspection systems

Sharing information on inspections and quality defects

Waiving batch testing of products on import into their territories.

MRAs are typically narrow in scope, for example, excluding vaccines or plasma-derived products. One example of an MRA is that the EU Member States will not duplicate inspections conducted by the FDA for human medicines (27).

Public Quality Standards (ICH Guidelines and USP Methods)

Augmenting the regulations are public quality standards or guidelines to which pharmaceutical companies generally adhere even though they are not considered regulations. Our overview (10) describes these quality standards and organizations, and we will comment further on ICH guidelines and USP compendial methods.

International Conference on Harmonization (ICH guidelines)

ICH brings together regulatory authorities of Europe, Japan, and the United States, as well as pharma experts, to discuss scientific and technical aspects of product registration. ICH issues guidance documents in Quality, Safety, Efficacy, and Multidisciplinary topics (QSEM) (28). For analytical development and quality control chemists working under GMP, the most referenced documents are the ICH quality guidelines (Q guidelines) listed in Table III.

USP is an independent, nonprofit organization whose mission is to improve world health by setting public standards to help ensure the quality, safety, and benefits of medicines and foods (29). They issue the USP-NF, a combination of two compendia, the United States Pharmacopeia and the National Formulary.

USP monographs contain tests, procedures, and acceptance criteria frequently employed to facilitate consistency in testing across the generic industry. Monograph testing requires the use of compendial reference standards. The USP also contains general chapters on standard techniques and general methodologies applied across many monographs used in specifications and certificates of analysis (CoA) (16–18). Also, the general chapters cover a broader scope, like method validation, verification, and method transfer. Validation of compendial methods is not required but must be verified for suitability for the intended use of the sample in your laboratory according to USP <1226>. The common USP methods often used in specifications and CoAs are listed in Table IV.

GMP Violations and Enforcement Actions by US FDA

If a company is not complying with cGMP, any drug it makes is considered “adulterated” under the law. This kind of adulteration means that the drug was not manufactured under conditions that comply with cGMP. Regulatory actions against companies with unacceptable cGMP compliance are intended to prevent the possibility of unsafe and ineffective drugs from getting to market (10,30). Observations found during inspections (GMP and pre-approval inspections [PAI]) by the FDA are formally documented using Form 483. Subsequent corrective actions by the company to address 483 observations can be quite involved. Repeated violations may result in the company receiving a warning letter from the FDA. Although the FDA cannot force a company to recall a drug, companies usually will recall voluntarily or at the FDA’s request. The FDA can warn the public and seize the drug in extreme cases. Seizure and injunction cases often lead to a court order and a subsequent “consent decree” that requires companies to take defined corrective steps to correct cGMP violations. Companies may be prohibited from distributing products until the consent decree is lifted (30).

Some Basic GMP Elements: The Five “P’s”

GMP regulations are formal, detailed, and complex. They include elements related to specific operations or business categories, such as manufacturing, process validation, or product recalls. However, some basic principles apply to every operation. Table V describes common elements of cGMP, sometimes called the “5 P’s”—people, processes, procedures, premises, and products (31).

GMP in Drug Development and QC Laboratories

Figure 2 shows an example of the organizational structure of quality systems and processes in a GMP manufacturing/ development facility, including those within a QC laboratory. Figure 2 was extracted from reference 10 and revised to add further components for GMP.

Regulatory agencies encourage companies to adopt a Quality by Design (QbD) approach to drug development (32), such that quality is designed into the entire manufacturing process rather than relying solely on QC testing at release. This is achieved by gaining product and process knowledge during development. Organizations will define a Quality Target Product Profile (QTPP) for the drug and use risk assessment techniques to identify Critical Quality Attributes (CQAs) to be monitored during production and at release. The analytical development chemist will then develop and validate methods using a multivariate DoE approach to execution and analysis to assess CQAs captured in product specifications for release testing (16,19,33–34). QbD also utilizes a lifecycle approach or continuous method improvement throughout the product lifecycle. For example, the method will be monitored over time to identify trends such as system suitability failures or unusually high numbers of deviations. The analytical chemist may redevelop and revalidate the method because of changes in process (such as for quantitation of new degradation products) or for method improvement purposes.

Phase-Appropriate GMP Compliance Approach

As described elsewhere (10,14,17), regulatory oversights increase throughout drug development. In late-phase development laboratories, analytical chemists develop and validate robust methods suitable for testing registration stability lots, releasing clinical trial materials (CTM) and transferring to manufacturing sites within the company or at a third party. Method validation attributes are described in ICH Q2 (R2), USP <621>, <1225>, and other references (16,19). Typical validation attributes include specificity, accuracy, linearity, range, precision (repeatability and intermediate precision), limits of detection and quantitation, robustness, and system suitability (including stability of standard and sample preparations) (19). As these late-phase procedures are intended to be transferred to QC units in the manufacturing facilities of commercial batches, procedures and records should be sufficiently detailed to ensure the transfer will be successful. Guidance for analytical method transfer studies is given in USP <1224>. Equipment in these labs must be fully qualified (IQ/OQ/ PQ) (16), and computerized systems must comply with 21 

Part 11 requirements for electronic records and signatures (8). This provides assurance the equipment is performing as intended and that data cannot be manipulated or erased.

There is no specific regulation on Good Documentation Practice (GDocP), though it is a central pillar of GMP compliance. GDocP includes the principles of “ALCOA+,” which stands for Attributable, Legible, Contemporaneous, Original, Accurate (plus Complete, Consistent, and Enduring) (35). These are integral to cGMP processes, as the main objective of reliable and traceable documentation is to establish, control, monitor, and record all activities that impact product quality. The term GDP is often used, though it should not be confused with Good Distribution Practices for manufacturing facilities.

A QC analyst is a laboratory scientist whose primary duties are to measure and test raw materials, active ingredients, in-process control and intermediates, excipients, and finished products at a manufacturing facility, contract manufacturing organization (CMO), or contract research organization (CRO). The QC analyst may be responsible for stability testing and participating in investigations of out-of-trend or out-of-specification results. Testing is controlled through general SOPs, work instructions, and analyte-specific test methods. The test method must be validated in a GMP laboratory using 21 

Part 11 compliant data systems (16,18). For example, OOS results and deviations are reported, investigated, and recorded. Their work is subject to inspection and audit since the analytical results they generate determine whether raw materials are dispositioned for use in compounding drug products or whether drug products comply with specifications for release and distribution to patients. Deficiencies found during inspections and audits lead to a CAPA to resolve the deficiency and avoid its recurrence.

The Restrictive GMP Work Environment for QC Analysts

Many laboratory scientists working under GMP are unhappy about the restrictive work environment in adhering to numerous SOPs and time-consuming documentation and review processes. In an ideal world, a quality system should be possible with efficient procedures for GMP compliance. Nevertheless, after reacting to deviations from audits and inspections, many companies’ quality systems and procedures have grown to be overly complex and bureaucratic with time. These inefficient procedures burden the daily routines of the QC analyst and prolong the overall drug development timeline. Pharmaceutical companies may benefit from a holistic re-evaluation of their internal policies and processes to identify opportunities to streamline workflows without impacting compliance.

cGMP regulations provide minimum quality standards to ensure quality is built into pharmaceuticals’ design and manufacturing process. The analytical development and QC analyst are essential to compliance by adhering to cGMP guidelines. Hence, their data are valid, traceable, and reproducible, ensuring that products are safe and efficacious.

This paper provides a high-level overview of cGMP regulations and related public quality standards for pharmaceutical scientists. Although the goals of GMP are straightforward, compliance activities are diversified and variable, driven by the company’s SOPs, resources, and compliance culture. The information presented here stems from health regulations, guidance documents, references, and the authors’ experience. Presenting a concise overview of this complex topic is challenging. The reader should refer to the original regulations and guidelines, references, and 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: Alice Krumenaker of Hovione, Anissa Wong of Gilead, David VanMeter of Sartorius, He Meng of Sanofi, Mike Shifflet of Kenvue, Kate Evans from Longboard Scientific, and Zedong Dong of Innovent Biologics.

(1) CFR Title 21 - Food and Drugs, Part 210 - 

Current Good Manufacturing Practice in Manufacturing, Processing, Packing, or Holding of Drugs, 

Good Manufacturing Practice for Finished Pharmaceuticals. 

Current Good Manufacturing Practice for Positron Emission Tomography Drugs, 

Good Manufacturing Practice for Human Food, 

Quality System Regulation (Medical Devices), 

Good Manufacturing Practice for Dietary Supplements, 

Electronic Records; Electronic Signatures, 

Application for FDA Approval to Market a New Drug, 

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

Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients

The Certified Pharmaceutical GMP Professional Handbook

Good Manufacturing Practices for Pharmaceuticals 

Fundamentals of Pharmaceutical and Biologics Regulations: A Global Perspective, 

Regulatory Affairs Professionals Soc. 2023.

Analytical Testing for the Pharmaceutical GMP Laboratory, 

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

(18) Kou, D.; Wigman, L.; Yehl, P.; Dong, M. W. Separation Science in Drug Development, Part 4: Quality Control, 

(19) Dong, M. W., Huynh-Ba, K., Wong, A. W. Validation of Stability-Indicating HPLC Methods for Pharmaceuticals: Overview, Methodologies and Case Studies, 

(20) Singh, N., Good Manufacturing Practices, online course at U. C. Santa Cruz Silicon Valley, Extension, 2024.

Drug Quality Fundamentals, Quality Control of Small Molecule Drugs and Recombinant Biologics

, Course at U. C. Santa Cruz Silicon Valley Extension, 2017.

(22) Doneski, L.; Roos, D.; Dong, M. W. Good Laboratory Practice: An Overview for the Analytical Chemist, 

(23) EudraLex - Volume 4 - Good Manufacturing Practice (GMP) guidelines, European Commission, 2022; 

https://health.ec.europa.eu/medicinal-products/eudralex/eudralex-volume-4_en

(24) World Health Organization, TRS 1044 - Annex 6: 

WHO good practices for research and development facilities of pharmaceutical products, 

International Harmonization via ICH, WHO, and other Global Initiatives in Fundamentals of Pharmaceutical and Biologics Regulations: A Global Perspective

(26) Pharmaceutical Inspection Co-operation Scheme (PIC/S), PE-009-17 (Parts I & II), Pharmaceutical Inspection Convention, 

(27) Questions & Answers on the impact of Mutual Recognition Agreement (MRA) between the European Union and the United States as of 31 May 2023, EMA, 

https://www.ema.europa.eu/en/documents/other/questions-answers-impact-mutual-recognition-agreement-between-european-union-united-states-31-may_en.pdf

(28) International Conference on Harmonization, 

https://www.ich.org/page/quality-guidelines

Column: Perspectives in Modern HPLC

We provide a high-level overview of Current Good Manufacturing Practice (cGMP) regulations and their impact on the analytical chemist in the pharmaceutical industry.

Current Good Manufacturing Practice (cGMP): An Overview for the Analytical Chemist

Enumerable types of gene therapies are collectively one of the fastest-growing areas of biopharmaceutical products today. Gene therapies are generally defined as treatments, perhaps even cures, for diseases through the transfer of genetic material to host cells. The analytical technologies that exist to characterize these novel therapies are rapidly advancing. In this column, we will briefly present some of the basic concepts related to this novel product class, specifically related to formulation and delivery, focusing on adeno-associated virus (AAV) formulations. We will then briefly discuss the analytical technologies and approaches most commonly used to characterize these products, specifically on the characterization of AAV-related products. We will briefly discuss what many consider the gold standard in analytical tools, analytical ultracentrifugation (AUC), and then specifically discuss liquid chromatography-mass spectrometry (LC–MS) tools and methods (such as the multi-attribute method, MAM) that are becoming more commonly used in characterization.

Gene therapy is a procedure that enables gene improvement by means of the correction of the altered (mutated) genes, or targets site-specific modifications that have therapeutic treatment as a target molecule (1). The most common vectors are viruses that have evolved a way of enveloping and delivering their genes to human cells. The most widely used gene transfer vectors in clinical trials are adenoviral vectors, retroviral vectors, adeno-associated viruses, and herpes simplex viruses (1). Around 800 approved gene therapy clinical trials worldwide have been conducted or are still ongoing; a large majority of these trials use a viral vector for gene transfer (drug delivery) (2).

Adeno-associated viruses (AAV) belong to the parovirus family, and AAV has emerged as one of the main vectors used in gene therapy due to many useful attributes, including a lack of pathogenicity and continuous maintenance of the viral genome (3). The AAV viral vector consists of a protein shell protecting a small, single-stranded DNA genome of approximately 4.7 kilobases (kb). Efficient replication and viral gene expression of the AAV genome requires co-infection with a second helper virus, such as adenovirus or herpes. The single-stranded genome contains three genes, Rep (Replication), Cap (capsid), and Aap (assembly). The coding sequences are flanked by inverted terminal repeats (ITRs) that are required for genome replication and packaging. The AAV capsid consists of 60 copies of three types of viral proteins (VPs), VP1, VP2, and VP3, assembled in a ratio of 1:1:10 (VP1/VP2/VP3). The three VPs are encoded by the cap gene, alternatively spliced from one mRNA. There are over 100 naturally occurring AAVs that have been isolated in humans and animals, which differentiate from each other based on their capsid components, cellular tropism, transduction efficiency, and immunogenicity. Within the same viral serotype, the VPs share a common protein sequence (3–6).

AAV delivery technologies have led to the development of two FDA-approved gene therapy products. Luxturna, an AAV2-based gene therapy for a rare, genetic form of blindness, was approved in 2017 (7), and Zolgensma, an AAV2-based gene therapy to treat spinal muscular atrophy (SMA), was approved in 2019 (8).

Development of scalable and robust manufacturing processes is necessary for gene therapy products that meet clinical and market demands and achieve cost of goods targets (4,5). AAV viral vector manufacturing is a complex process that necessitates new approaches to meet the challenges of stability, safety, efficacy, quality, preventing degradation of AAV vectors during manufacturing, handling, and storage as well as compiling with the current Good Manufacturing Practice (cGMP) (4,5). Viral vector manufacturing workflows include upstream, downstream, formulation, and fill or finish processing unit operations as shown in Figure 1.

Unit operations in upstream viral vector production include: (i) plasmid development and production that requires a cis-plasmid, a trans-plasmid (that encodes the AAV rep and cap genes), and a helper plasmid (that encodes adenovirus [Ad] helper genes, E2A, E4, and VA RNA); (ii) cell expansion where E1 transduced cells (such as adherent HEK293T [a human embryonic kidney cell line that is typically used for AAV manufacturing] or suspension HEK293) are grown to a desired cell density; (iii) plasmid transfection where plasmids are introduced into cells; and (iv) viral vector production where transiently transfected cells produce the virus. While transient plasmid DNA transfection is adaptable to new AAVs and produces high particle yields, large amounts of plasmid DNA are needed to scale up for larger vector batches (4,9,10).

The downstream process includes methods for purification of viral particles from process- and product-related impurities, including host cell material, plasmid DNA, and empty capsids. A typical downstream process includes: (i) cell lysis; (ii) removal of nucleic acid; (iii) centrifugation or microfiltration to remove cell fragments and debris; (iv) affinity chromatography; (v) separation of full gene-containing infectious viruses from empty, non-infectious viruses, using cesium chloride gradient ultracentrifugation procedures or ion-exchange chromatography (IEC), and (vi) further purification by reduction of host cell proteins using core-bead adsorbents (4,9,10).

The AAV product manufacturing process can introduce many impurities throughout the manufacturing process such as (but not limited to) non-encapsulated DNA, empty capsids, aggregated capsid, and residual proteins, and some of these are concerns for immunotoxicity (11).

The major challenge of the AAV manufacturing process is production of capsids that are not packaged with the therapeutic transgene and are therefore referred to as empty capsids. The empty capsids are considered as a product-related impurity, and could potentially cause impairment of potency through receptor competition. Along with empty capsids, partially filled capsids are also produced during the manufacturing process and may contain packaged process-related impurities or truncated genomes. This necessitates the use of analytical techniques that can provide information regarding the content distribution of AAV capsid content (12).

Several studies have evaluated analytical methods for their ability to assess capsid content, as the measurement of AAV product is extremely challenging. These methods include density gradient separation using analytical ultracentifugation (AUC), cryo-electron microscopy (cryo-EM), liquid chromatography-mass spectrometry (LC–MS), size exclusion chromatography coupled to multiangle light scattering (SEC-MALS), capsid titre-to-genome titre ratio using q-PCR, and anion-exchange chromatography (AEX) (4,12–14).

Analytical ultracentrifuge (AUC) is a high-resolution technique and has become the gold standard for AAV quantification (15–17). It can be used to analyze capsid content through the difference in buoyant density due to differences in mass (15–17) between empty and full capsids.

AUC measures the concentration gradients of sedimenting molecules during the application of a centrifugal force while maintaining temperature and rotational speed. It is equipped with the interferometry optical system that allows the determination of concentration gradients for macromolecules in solution. Full capsids have a sedimentation coefficient c(s) of approximately 105 s, whereas empty capsids have values of approximately 66 s. As full capsids have a greater buoyant density than empty capsids, they sediment more quickly through solution (18).

A single sedimentation velocity run on an adenovirus sample can be used to detect and quantify different forms of virus particles and subviral particles. These forms include (i) intact virus monomer particles; (ii) virus aggregates; (iii) empty capsids (ECs); and (iv) smaller assembly intermediates or subparticles formed during normal or aberrant virus assembly. The data can be obtained by direct boundary modeling of the AUC data generated from refractometric or UV detection systems to provide characterization details (18–20). However, the major challenge for the AUC method is the requirement of large quantities of material along with a low throughput.

Mass spectrometry (MS) has emerged as a high-resolution analytical technique in protein characterization. LC–MS offers a robust method to directly measure the protein molecular weight of the AAV capsid, providing both specificity and sensitivity for rapid confirmation of capsid serotypes. Peptide mapping methods for different AAV capsids (including chemically engineered capsids) have been optimized for high sequence coverage and precise amino acid structure identification using tandem mass spectrometry (MS/ MS), and are being used for capsid protein characterization and process impurity identification (21), as shown in Figure 2.

Intact AAV Capsid Characterization Using LC–MS

A unique application of MS is the intact AAV analysis and its ability to differentiate fully filled (capsids containing the genes of interest) versus partially filled or empty capsid particles also referred to as the content ratio. The capsid of each AAV serotype consists of 3 VP proteins: VP1 (82kDa), VP2 (66kDa), and VP3 (60kDa), which share an overlapping open reading frame. Analytical LC–MS and LC-coupled tandem MS (LC–MS/MS) provide specificity, sensitivity, and speed in analysis in less than 5 min. Intact LC–MS can be used to determine the exact mass gain for capsid chemical modification, thus providing information of the AAV capsid (21,22).

Compared with other methods such as AUC, SEC, and IEC, native MS has the advantages of minute sample requirements, rapid analysis, high sensitivity, and high mass resolution to distinguish between full, partially full, and empty capsids. However, one of the main challenges in native MS analysis of AAV vectors is poor resolution of viral particles containing variable lengths of the genomic content (22).

Peptide Mapping Analysis of AAV Capsids using LC–MS/MS 

Characterization of AAV viral proteins includes the evaluation of capsid stoichiometry, identity, and post-translational modifications (PTMs). These parameters of the viral capsid are the critical quality attributes that can influence the tropism, infectivity, and transduction efficiency of the AAVs (21). LC–MS/MS is one of the most valuable and advanced tools in analytical chemistry to conduct a comprehensive peptide map analysis including post-translational modifications as it is high-throughput, amenable to quality control, has great specificity, and speed to ensure the quality and safety (contaminants in each AAV batch will be detected in LC) of the final vector product. Coupling MS/MS further allows to peptide map the precise amino acid sequence of each capsid protein, including chemical conjugations, and the chemistry of the AAV capsid proteins. Peptide mapping analysis of the AAV capsid proteins has been shown to achieve high sequence coverage and precise amino acid structure elucidation, including the identification of covalent modifications (22).

While the separation of VP3 is achieved, distinguishing VP1 and VP2 is challenging as they share a common C-terminal sequence. Integrating a multi-attribute characterization of AAVs using a two-dimensional liquid chromatography (2D- LC) coupled with MS can be used to perform comprehensive and high-throughput analysis. This approach allows for different chromatography techniques to be coupled with MS for analytical characterization of biomolecules (23). The multi-attribute method (MAM) is a LC–MS-based method that is used to characterize and monitor product quality attributes (PQAs) at the amino acid level of a biopharmaceutical product. MAM relies on proteolytic digestion of the sample followed by reversed-phase chromatographic separation and high-resolution LC–MS analysis in two phases (24). There are two aspects of the MAM workflow—first, a discovery study to determine quality attributes for monitoring. This involves the creation of a targeted library based on high-resolution retention time along with an accurate mass analysis. The second aspect of MAM is the monitoring phase that relies on the target peptide library and new peak detection. It uses differential analysis of the data to determine the presence, absence, or change of any species that may affect the stability of the biotherapeutic.

The protocol consists of three main stages:

, or proteolytic digestion of the protein.

Reversed-phase chromatographic separation of the generated peptides and detection.

 During Phase I of MAM (discovery phase), data-dependent acquisition (DDA) MS/MS is performed to enable confident identification of peaks and development of a peptide library. During Phase II of MAM (monitoring phase), full MS acquisition is carried out for the monitoring of predefined PQAs or CQAs.

Data processing, analysis, and reporting for each of the two phases.

Recently, a 2D-LC–MS platform that combined anion-exchange chromatography (AEX) and RPLC–MS was used to measure the amounts of empty and full capsids, characterize viral proteins, and evaluate capsid identity and PTMs. High-resolution separation in the first dimension was achieved using AEX, with a salt gradient to achieve empty and full capsid separation. Using the heart-cutting technique in 2D-LC, peaks of interest were then transferred and stored in different trapping loops. The fractions were sequentially transferred to second-dimension LC, where they were subjected to online denaturation and desalting in a trap column. The AAV capsid was denatured by online acidification, and the MS-incompatible salt was then removed. VPs were separated by RPLC in the second dimension, followed by MS analysis (23).

The main advantage of the method is the high-throughput and multi-attribute characterization of the sample in a single experiment. The method demonstrates the ability to perform online fractionation and subsequent downstream analysis in an integrated manner. Integration of additional methods will enable multi-attribute characterization in a single experiment with minimal sample handling required for different AAV serotypes (24). MAM can be applied throughout the product life cycle, from process development through upstream to downstream processes up to quality control release, and can be used to enforce current Good Manufacturing Practice regulations.

(1) Cavazzana-Calvo, M.; Thrasher, A.; Mavilio, F. The Future of Gene Therapy. 

https://clinicaltrials.gov/ct2/results?cond=&ter m=AAV&cntry=&state=&city=&dist=.

(3) Wang, D.; Tai, P. W. L.; Gao, G. Adeno-Associated Virus Vector as a Platform for Gene Therapy Delivery. 

 358–378. DOI: 10.1038/s41573-019-0012-9

(4) Srivastavaa, A.; Mallelab, K. M. G.; Deorkara, N.; Brophy, G. Manufacturing Challenges and Rational Formulation Development for AAV Viral Vectors. 

 2609−2624. DOI: 10.1016/j.xphs.2021.03.024

(5) Wright, J. Manufacturing and Characterizing AAV-Based Vectors for Use in Clinical Studies. 

(6) Samulski, R.J.; Muzyczka, N. AAV-Mediated Gene Therapy for Research and Therapeutic Purposes. 

 (1), 427–451. DOI: 10.1146/annurev-virology-031413-085355

https://www.fda. gov/vaccines-blood-biologics/cellular-gene-therapy-products/luxturna (accessed 09-29-2023).

We present formulation and delivery of gene therapy products, focusing on adeno-associated virus (AAV) formulations. We also discuss the analytical technologies most used to characterize these products.

November 2023 | LCGC North America

The Evolution of LC Troubleshooting: Strategies for Improving Peak Tailing

Molecular Spectroscopy: Underutilized Detection for Gas Chromatography

Current Good Manufacturing Practice (cGMP): An Overview for the Analytical Chemist

Characterization of Adeno-Associated Virus (AAV) Gene Therapy Products

Simultaneous Determination of 17 Residual Monomers in Adhesives by Gas Chromatography–Mass Spectrometry