Pharmaceutical Analysis

Supercritical fluid chromatography (SFC) is a well-established analytical technique used in the pharmaceutical industry for decades. However, it is still considered a new technique in some areas, for example, implementing the technique for purity profiling in late-stage development and production. In pharmaceutical analytical departments, SFC serves a wide variety of purposes, including compound purification, purity profile, and chiral analysis. Depending on the phase of drug development, the analytical performance requirements, such as speed of analysis, efficiency, and sensitivity, may vary considerably. The end goal is to provide robust, reliable and transferable analytical SFC methods. The challenges for future development and widespread implementation of SFC and the implementation of SFC in quality control (QC) laboratories using modern instrumentation are also discussed.

These ten propositions are widely acknowledged, but frequently neglected, by practitioners of high performance liquid chromatography (HPLC).

Researchers from Merck and Agilent Technologies have developed a simple and fast generic gas chromatography–flame ionization detection (GC–FID) method for the quantitation of volatile amines in pharmaceutical drugs and synthetic intermediates.

Comprehensive two-dimensional liquid chromatography (2D-LC) was used for detailed profiling of various nonionic ethoxylated surfactants applied in pharmaceutical formulations. Hydrophilic-interaction chromatography (HILIC) and reversed-phase liquid chromatography (LC) were used as the first and second dimensions, respectively. Detection was performed with evaporative light-scattering detection (ELSD) for general profiling and with single-quadrupole mass spectrometry (MS) for structure elucidation of individual peaks and for class-type confirmation of peak-groups.

With the growing use of illegal opioids, analysts should be prepared for a large influx of samples in their laboratories. While the workload may be increasing, the number of analysts and equipment may not, so the need for faster and better liquid chromatography–mass spectrometry (LC–MS) methods is important. By implementing an efficient sample preparation technique for matrix cleanup for some of the most common and traditional opioid matrices-blood and urine-coupled with a rapid and accurate LC method, laboratories can address the analytical needs for this growing problem.

The drug discovery process can be accelerated by chromatographic profiling of analogs by measuring their nonspecific binding to proteins and lipids and then by modelling in vivo distribution. A balanced potency and chromatographically determined membrane and protein binding ensure the selection of compounds with the highest probability to show the desired in vivo distribution behaviour for efficacy and reduced toxicity. The first part of the article will discuss the high performance liquid chromatography (HPLC)-based measurements of lipophilicity and biomimetic properties, while the second part will discuss the models derived from the measured data of known drug molecules and drug discovery compounds.

Chromatographic method development for pharmaceutical analysis can benefit from in silico steered serial coupling of column segments containing different stationary phases of varying length. Contrary to column coupling through trial and error, in stationary-phase optimized selectivity (SOS)-based chromatography the retention of all solutes is predicted for all possible column combinations allowing a rational selection of the optimal column combination. The possibilities of the strategy now surpass the initial usage in isocratic high performance liquid chromatography (HPLC) on dedicated commercial column segments, and allow applications in gradient-, green-, preparative-, and in supercritical fluid chromatography (SFC) on conventional column hardware. Current possibilities, pharmaceutical applications, a downloadable algorithm, and weaknesses of the approach are discussed to allow broader implementation of this methodology in separation science.

Significant recent advances now enable routine usage of HDX-MS for comparing the conformations of biopharmaceutical products.

HIC analysis is carried out using a reversed salt gradient, starting with a high salt concentration and moving to low salt concentration to facilitate protein elution.

Chromatographic techniques with mass spectrometric detection are important enablers in modern drug discovery. With the development of robust instrumentation and implementation of user-friendly software (or software packages), non-expert users can now walk up to easily accessible advanced chromatographic systems and perform experiments at their own convenience. Although remarkable improvements in robustness and ease-of-use have happened since the introduction of the first high performance liquid chromatography–mass spectrometry (HPLC–MS) systems, the instrument performance still needs to be qualified and monitored to ensure consistent high-quality results. This article will demonstrate how a simple test mixture of carefully selected compounds can facilitate both the development of generic ultrahigh-pressure liquid chromatography–mass spectrometry (UHPLC–MS) methods and automated performance monitoring of multiple instruments located in separate laboratories and buildings.

All agencies have issued varying guidances for the approval of recombinant, biosimilars of biopharmaceuticals. However, their impact or meaning is in our understanding and that all submittals are considered on a case-by-case basis.

Quantitative determination of the counterions associated with pharmaceutical salts is a mandatory requirement for quality control. While ion chromatography (IC) is the standard technique in most laboratories, capable of delivering excellent sensitivity, specificity and flexibility, there are other simpler and quicker analytical methodologies that may should be considered for this quality control application.

In this article, we discuss the use of CE-MS (sheath flow interface) for analysis of intact proteins as well as of protein digests. We discuss the unique aspects that the user needs to be aware of while testing biotherapeutics versus small molecule drugs. We also highlight that the optimization of CE and MS parameters together result in the creation of a more robust and reproducible protein analysis approach. Finally, we list some of the most common errors that are likely to occur during CE-MS analysis and suggest ways to overcome them.

There can be significant benefits by standardizing HPLC columns in a pharmaceutical development laboratory. Here is a story of how one organization attempted to encourage its staff to develop HPLC methods using fewer column brands and dimensions to reduce waste and efforts in method transfers downstream.

In this column, we introduce the basics of today’s approaches for doing intact protein dissociation with mass spectrometry (MS), or top-down sequencing (that is, rather than the more conventional peptide-based “bottom-up” sequencing where future improvements might occur, advantages and limitations of using top-down sequencing, possible applications, and why it has become such an important and pursued research area for many.

A universal generic HPLC or UHPLC method with a primary modern column that works well for most drug analyses in a few minutes would be an attractive idea for many laboratories. With advances in column technologies, this ideal scenario is becoming more realistic, as demonstrated in the proposed 2-min generic method shown here. In addition, rationales for the selection of column and operating conditions are discussed, together with ways to extend this generic method as a starting point for stability-indicating applications by simple adjustments of gradient time and range.

Subcritical water chromatography (SBWC) is seen as an increasingly enticing prospect to replace high performance liquid chromatography (HPLC) in industrial settings because of its low costs and environmental impact. Yu Yang of East Carolina University, in Greenville, North Carolina, USA, has conducted research into this process for two decades. He recently spoke to LCGC about his work, why companies should consider SBWC, and its role in pharmaceutical analysis.

Glycosylation of monoclonal antibody (mAb) therapeutics is widely recognized by the regulators and the industry as a critical quality attribute (CQA). Hence, it is necessary that glycosylation is measured and adequately controlled during production. This installment reviews the various process parameters and raw material attributes that affect glycosylation, as well as the different analytical tools that are used for characterization, with greater emphasis on the chromatographic methods of analysis. Key recent advancements that have occurred in the past five years are also discussed briefly. While significant progress has been made in the monitoring of glycosylation, its real time control has yet to be demonstrated.

This fourth and last installment in the series of “Separation Science in Drug Development” provides an overview of modern practices of Quality Control in small molecule drug development including activities such as setting specifications, method validation/transfer, release and stability testing, and authoring CMC sections of regulatory filings.

The third installment in this series provides an overview of modern practices of separation science in small-molecule drug development. It highlights approaches in HPLC method development and physical/chemical characterization to support process chemistry and formulation development, and for assessment/control of the clinical trial materials. The role of the separation scientist in analytical development and salient chromatographic methodology trends are discussed.

A systematic approach for formation of sigmoidal gradient for analysis of biopharmaceutical proteins has been reviewed and discussed together with two case studies. The first application involves HPLC separation of the various product related variants of a microbial expressed biotherapeutic, granulocyte colony stimulating factor (GCSF). The second case study involves separation of the charge heterogeneity related variants for a monoclonal antibody (mAb) biotherapeutic product. In all cases, it is observed that the use of sigmoidal gradient successfully reduces the analysis time significantly (from 70 minutes to 15 minutes for GCSF and from 40 minutes to 4 minutes for the mAb) while retaining the selectivity and the resolution.

The United States Food and Drug Administration (FDA) has issued a new guidance document, Analytical Procedures and Method Validation for Drugs and Biologics. The guidance is quite general in nature. Anyone hoping for specific recommendations on topics such as which methods to use will be disappointed. Industry experts say that i really isn’t feasible for the FDA to provide detailed recommendations about analytical methods for biopharmaceuticals, however.

This installment provides an overview of high-throughput characterization techniques of drug leads to support small molecule drug discovery programs in a pharmaceutical company. A myriad of analytical chemistry techniques including separation science methodologies are used to confirm the structures and identities, quantitating the concentrations of stock solutions, and measuring key physicochemical properties of the new chemical entities (NCE). A case study is used here to illustrate the details of these applications in high-throughput characterization.

Here in part II of our series on assessing protein aggregation, we provide an overview of best practices for achieving this goal, including the importance of using a multimethod approach.Here in part II of our series on assessing protein aggregation, we provide an overview of best practices for achieving this goal, including the importance of using a multimethod approach.

This installment provides an overview of modern practices of high-throughput purification to support small-molecule drug discovery.

Benedetto Natalini of the University of Perugia, Italy, spoke to Bethany Degg of The Column about the driving forces in pharmaceutical analysis, including the importance of regulation, chirality, and miniaturization.

Experimental results and method validation data from two analytical case studies of complex small?molecule pharmaceuticals illustrates the utility and advantages of UHPLC in high-resolution separations.

Most analytical approaches used for the determination of low-molecular-weight noncovalent aggregates in protein pharmaceuticals suffer from important limitations. This first part of this two-part column series discusses those limitations.

In this installment, we discuss fluorescence-detection size exclusion chromatography (FSEC) technology and its implications in protein characterization, specifically the development and characterization of biologics.  

A discussion of the role of the gradient in separation by reversed-phase and ion-exchange HPLC, with data from two products to illustrate the key points