Guest editors Valentina D’Atri from the University of Geneva in Switzerland and Jelle De Vos from the Research Institute for Chromatography (RIC) in Belgium, interviewed leading luminary of biopharmaceutical analysis, Reed Harris on critical quality attributes (CQAs), contemporary trends in biopharmaceutical characterization, and how he earned the moniker “Dr. Doom.”
The 51st International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2023), chaired by Michael Lammerhofer and Oliver J. Schmitz, was held from 18-22 June in Dusseldorf, Germany. This instalment of "Column Watch" presents many of the highlighted topics and trends observed at this exceptionally well-run symposium.
A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with a dual electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) source was developed for analyzing 102 pesticides and five mycotoxins that are regulated by the state of Colorado in hemp. The limit of quantitation (LOQ) of the 102 pesticides and 5 mycotoxins met Colorado state action limit requirements for these analytes in a hemp matrix. For this study, 88 out of 102 pesticides and all 5 mycotoxins were analyzed using LC-MS/MS with an ESI source, whereas the remaining 14 pesticides were determined using LC-MS/MS with an APCI source. A simple, fast, green, and cheap acetonitrile solvent extraction method was used to extract the pesticides and mycotoxins from the hemp matrix with good extraction efficiency in the range of 80-120%. A hemp matrix is challenging and causes matrix effects such as ion suppression or enhancement. The LC method was optimized and 30 internal standards were added to reduce and compensate for these matrix effects to obtain method accuracy in the range of 70-120%. The ionization mechanism of nonpolar pesticides (normally analyzed by gas chromatography-tandem mass spectrometry [GC-MS/MS]) with an APCI ion source was elucidated.
“Questions of Quality” is 30 years old! What, if anything, has changed in chromatography laboratories over that time? What, if anything, remains the same?
Therapeutic mRNA is receiving growing interest in various therapeutic applications such as genome editing, cancer immunotherapy and prophylactic vaccines. As with other drugs, it is essential to guarantee product quality. Among the critical quality attributes of therapeutic mRNA, characterization of the capping and poly(A) tail are of the greatest importance because of their involvement in mRNA stability and in the efficiency of protein synthesis. This article presents a method for the simultaneous characterization of both attributes in a single sample preparation workflow. The method involves lipid extraction, various RNAse enzymes, purification steps and LC–MS to analyze the capping and poly(A) tailing.
Analytical methods that allow separation and identification of therapeutic proteins under native conditions play a crucial role in studying their higher-order structures and structure–function relationships. Recently, hyphenated techniques that combine native-mode separation with native mass spectrometry (nMS) have emerged as highly valuable tools for the targeted assessment of these quality attributes. This article outlines current native separation strategies coupled with nMS designed to characterize biopharmaceuticals close to their natural state. The methods provide worthwhile insights into aspects like aggregation, charge variants, conjugate stoichiometry, affinity, and conformation. As multidimensional chromatographic techniques and ion-mobility spectrometry become more accessible in laboratories, further advances in the development of native hyphenated techniques capable of simultaneously providing compositional, structural, and functional information on biopharmaceuticals can be expected.
Messenger ribonucleic acids (mRNA) therapeutics are becoming more widespread pharmaceutical tools to treat a wide range of diseases or infections, as highlighted by regulatory approval of two vaccines for SARS‑CoV-2. Alongside their use as vaccines, they also play a role in protein replacement therapy to ensure therapeutic protein is synthesized within the patient. Structural elements, such as the 5’ cap, UTR regions, reading frame, and poly A tail are considered as critical quality attributes (CQAs) that are subject to a range of analytical techniques. However, chromatography and other separation methods are commonly used for characterization and quantification of the drug substance and drug product. This article reviews a range of techniques available for separative analysis of mRNA therapeutics, their associated impurities, and delivery vehicles.
Anneli Kruve and Pilleriin Peets from Stockholm University in Sweden, discuss their latest research in machine learning and nontargeted liquid chromatography–mass spectrometry (LC–MS) to assess ecotoxicity.
Retention and selectivity in gas chromatography (GC) occur because of the thermodynamics of the phase transition from the vapor phase following sample injection to the solution phase of being dissolved in the stationary phase. Analytes then move along the column because the vapor phase is flowing. In this installment, we quickly review the basic thermodynamics underlying separation in GC, and we see how this impacts selectivity and retention. Last month, we saw how seemingly large differences in column polarity may still generate little, or even the opposite, effect on retention and generate confusion and difficulty in method development. This month, we explore this challenge more in-depth using thermodynamics and a freely available online GC simulator.
The primary analytical challenge is to selectively extract the target analytes using a suitable sample preparation technique and introduce them into the downstream analytical instrument. The critical step in the chemical analysis is sample preparation. Sorptive sample preparation techniques are among the new generation of microextraction approaches, and are compliant with green analytical chemistry principles. A recent intercontinental collaboration between two academic research laboratories—the Aristotle University of Thessaloniki, Greece, and the Florida International University, USA—has yielded a significant number of analytical/bioanalytical methods using fabric phase sorptive extraction (FPSE), magnet integrated fabric phase sorptive extraction (MI-FPSE), and capsule phase microextraction (CPME) for the isolation of various analytes from different complex sample matrices. A brief description of these techniques with regards to principle, synthesis, applications, and advantages and disadvantages along with paradigms is presented.
In our September installment of "Sample Prep Perspectives," we shared sample exam questions from our graduate Chromatography and Separations course as a refresher of some fundamental principles and to give readers the opportunity to test their knowledge. We've dusted off our files and found additional questions for this month's column. These questions tend to focus on dealing with octanol-water partition coefficients, as well as more practical applications of sample preparation.
Partition coefficients, log P or Kow, are often used in introducing and describing new extraction techniques. They can also be used in developing new separation methods. An understanding of partition coefficients, along with diffusion, is necessary to more completely guide separation processes. As with the application of any theoretical concepts, several assumptions may be applied. This month, we take a dive into the practical applications of partition coefficients as they are commonly applied to chemical extractions.
A recent trend in the design of liquid chromatography (LC) instrumentation is the move towards miniaturized and portable systems. These smaller platforms provide wider flexibility in operation, with the opportunity for conducting analysis directly at the point of sample collection rather than transporting the sample to a centralized laboratory facility. For the manufacturing of pharmaceutical and biopharmaceutical products, these platforms can be implemented for process monitoring and product characterization directly in manufacturing environments. This article describes a portable, miniaturized LC instrument coupled to a mass spectrometer (MS) for characterization of a biopharmaceutical monoclonal antibody (mAb).
This article covers liquid chromatography (LC) columns and accessories commercially released after Pittcon 2022 through the 2023 conference held in Philadelphia, USA. As in the past, LCGC sent out a survey in late 2022 and early 2023 asking vendors to supply information on products launched over the past year. Note that new products for gas chromatography (GC), LC instrumentation and software, and sample preparation are covered elsewhere. Information for this article was obtained over the course of many months; thus, it is possible that some information has been missed or misinterpreted. The reader is encouraged to check with specific vendor sites for additional product releases as well as more detailed information on product usage and attributes. Links to vendor sites are provided where applicable.
Phytotoxins, the secondary metabolites synthesized by plants, are now recognized as a new category of environmental micropollutants. So far, only a limited number of phytotoxins have been detected and reported in terrestrial and aquatic environments, partially due to the analytical challenge involved. In this article, a robust, reliable, and efficient approach, namely Source Supported Suspect Screening (4S), is introduced for high-throughput analysis of phytotoxins from their plant origin to the downstream environmental compartments. The approach was established on an ultrahigh-performance liquid chromatography–electrospray ionization–quadrupole‑time‑of‑flight-mass spectrometry (UHPLC–ESI-QTOF-MS) analytical platform where optimal sample preparation and chromatographic conditions were investigated and a new suspect screening protocol was suggested.
Since the publication of the first tenets of green chemistry about 25 years ago, the conversation about sustainability and chemistry has slowly grown, and today seems at a fever pitch, as companies are adding sustainability officers, universities are adding curriculum, and chemistry-related processes being updated regularly. In this installment, we explore the impact of green chemistry on analytical chemistry and gas chromatography. We define green chemistry, and discuss what it is and what it is not. For example, it is not "chemistry lite." We then briefly review relevant literature on green analytical chemistry, and begin a conversation on green gas chromatography and the many areas in which gas chromatography-based methods can be made "greener.
Detectors in gas chromatography (GC) are unique in all analytical science. The detector performs several difficult functions that make sensitive and selective analysis possible. In this installment, we will explore the challenges involved in detecting the effluent from a capillary gas chromatographic column, and summarize the many detector options. We will then consider basic ideas and tasks that allow detectors to function properly. Finally, we will examine some specific troubleshooting and preventative maintenance tips for common detectors. We will see that, with some simple preventative thinking and action, detectors in gas chromatography can provide excellent sensitivity and selectivity for years of use.
Molecular spectroscopy techniques, most commonly ultraviolet-visible (UV-vis), are usually thought of as analyzing liquid-phase 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.
