Stronger together: Analytical techniques for recombinant adeno associated virus.

With recent FDA approval of two recombinant adeno-associated virus (rAAV)-based gene therapies, these vectors have proven that they are suitable to address monogenic diseases. However, rAAVs are relatively new modalities, and their production and therapy costs significantly exceed those of conventional biologics. Thus, significant efforts are made to improve the processes, methods, and techniques used in manufacturing and quality control (QC).

Superheated reversed phase chromatography with ultrashort columns for the analysis of therapeutic proteins.

Mild or elevated temperatures are routinely used for the analysis of therapeutic proteins by reversed phase liquid chromatography. Generic conditions can be used for the analysis of monoclonal antibodies, and may be adapted for species derived thereof, for instance their immuno-conjugates. Beyond platform monoclonal antibodies, many novel, non-covalent protein complexes are also frequently pursued as protein therapeutics.

Use of Ultrashort Columns for Therapeutic Protein Separations. Part 1: Theoretical Considerations and Proof of Concept.

Due to the particular elution mechanism observed with large solutes (e.g., proteins) in liquid chromatography, column length has less impact in controlling their retention compared to small solutes.

Protocols for the analytical characterization of therapeutic monoclonal antibodies. III - Denaturing chromatographic techniques hyphenated to mass spectrometry.

The full analytical characterization of therapeutic monoclonal antibodies (mAbs) requires a large variety of complementary information that can be obtained by chromatographic methods. A series of protocols papers has been proposed to cover the chromatographic techniques and the enzymatic and chemical sample preparation procedures generally applied for the analytical characterization of therapeutic mAbs. The present protocol paper focuses on denaturing chromatographic techniques hyphenated to mass spectrometry, namely reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC), to assess the subtle mAbs structural heterogeneity resulting from glycosylation patterns and post-transcriptional modifications (PTMs).

Characterizing various monoclonal antibodies with milder reversed phase chromatography conditions.

Reversed phase liquid chromatography (RPLC) of therapeutic monoclonal antibodies (mAbs) is often performed at elevated temperatures (80-90 °C) and in the presence of relatively high concentrations of TFA (0.1%). Under such conditions, it is possible to achieve suitable performance in terms of peak shapes and recoveries.

Simultaneous determination of ten nonsteroidal anti-inflammatory drugs from drinking water, surface water and wastewater using micro UHPLC-MS/MS with on-line SPE system.

A simple, accurate and sensitive micro UHPLC-MS/MS method was developed and validated for the simultaneous determination of 10 nonsteroidal anti-inflammatory drugs (NSAIDs) from different environmental matrices. The micro LC ‒ on-line SPE method described in this study allowed to determine the selected drugs at ultra-trace levels without the most commonly used complex off-line SPE sample preparation procedures. The presented method is capable of reaching satisfactory low LOQ values with analysing the sample directly after being diluted with water.

New developments and possibilities of wide-pore superficially porous particle technology applied for the liquid chromatographic analysis of therapeutic proteins.

This review paper discusses the success of columns packed with superficially porous particles (SPP) in liquid chromatography for the analysis of peptides and proteins. First, it summarizes the history of SPP, including the development of different SPP generations from particles of 50 μm to sub-2 μm. It also critically discusses the improved kinetic performance of SPP particles in comparison to fully porous particles.

Utility of a high coverage phenyl-bonding and wide-pore superficially porous particle for the analysis of monoclonal antibodies and related products.

A wide-pore silica-based superficially porous material with a high coverage phenyl bonding was evaluated for the analysis of monoclonal antibodies and antibody-drug conjugates. This new material is based on 2.7 μm particles having a shell thickness of 0.

Simultaneous determination of thirteen different steroid hormones using micro UHPLC-MS/MS with on-line SPE system.

Ultratrace analysis of sample components requires excellent analytical performance in terms of limits of quantitation (LOQ). Micro UHPLC coupled to sensitive tandem mass spectrometry provides state of the art solution for such analytical problems. Using on-line SPE with column switching on a micro UHPLC-MS/MS system allowed to decrease LOQ without any complex sample preparation protocol.

Determination of isoelectric points and relative charge variants of 23 therapeutic monoclonal antibodies.

Despite the popularity of therapeutic monoclonal antibodies (mAbs), data relative to their ionic physico-chemical properties are very scarce in the literature. In this work, isoelectric points (pIs) of 23 Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved mAbs were determined by imaged capillary isoelectric focusing (icIEF), and ranged from 6.1 to 9.

Current possibilities of liquid chromatography for the characterization of antibody-drug conjugates.

Antibody Drug Conjugates (ADCs) are innovative biopharmaceuticals gaining increasing attention over the last two decades. The concept of ADCs lead to new therapy approaches in numerous oncological indications as well in infectious diseases. Currently, around 60 CECs are in clinical trials indicating the expanding importance of this class of protein therapeutics.

Protocols for the analytical characterization of therapeutic monoclonal antibodies. II - Enzymatic and chemical sample preparation.

The analytical characterization of therapeutic monoclonal antibodies and related proteins usually incorporates various sample preparation methodologies. Indeed, quantitative and qualitative information can be enhanced by simplifying the sample, thanks to the removal of sources of heterogeneity (e.g.

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach.

Hydrophilic interaction liquid chromatography (HILIC) is a well-established technique for the separation and analysis of small polar compounds. A recently introduced widepore stationary phase expanded HILIC applications to larger molecules, such as therapeutic proteins. In this paper, we present some generic HILIC conditions adapted for a wide range of FDA and EMA approved recombinant monoclonal antibody (mAb) species and for an antibody-drug conjugate (ADC).

Protocols for the analytical characterization of therapeutic monoclonal antibodies. I - Non-denaturing chromatographic techniques.

Size-, charge- and hydrophobicity-related variants of a biopharmaceutical product have to be deeply characterized for batch consistency and for the assessment of immunogenicity and safety effects. Size exclusion chromatography (SEC) and ion exchange chromatography (IEX) are considered as the gold standard for the analysis of high molecular weight species (HMWS) and charge-related variants, respectively. Hydrophobic interaction chromatography (HIC) has drawn renewed attention to monitor the small drug payload distribution in the cysteine-linked antibody-drug conjugates (ADC).

Optimization of non-linear gradient in hydrophobic interaction chromatography for the analytical characterization of antibody-drug conjugates.

The goal of this work was to evaluate the potential of non-linear gradients in hydrophobic interaction chromatography (HIC), to improve the separation between the different homologous species (drug-to-antibody, DAR) of commercial antibody-drug conjugates (ADC). The selectivities between Brentuximab Vedotin species were measured using three different gradient profiles, namely linear, power function based and logarithmic ones. The logarithmic gradient provides the most equidistant retention distribution for the DAR species and offers the best overall separation of cysteine linked ADC in HIC.

Separation of Isomers of JWH-122 on Porous Graphitic Carbon Stationary Phase with Non-Aqueous Mobile Phase Using Intelligent Software.

Cannabimimetic compounds have gained an increasing attention from the forensic community during the past few years. The present study was aimed to develop a liquid chromatographic separation method for the analysis of JWH-122 and its methyl isomers. In Hungary, JWH-122 is scheduled as a narcotic compound and its methyl isomers fall into the new psychoactive substance category, attracting significantly milder punishment than JWH-122 does.

Impact of organic modifier and temperature on protein denaturation in hydrophobic interaction chromatography.

The goal of this study was to better understand the chromatographic conditions in which monoclonal antibodies (mAbs) of broad hydrophobicity scale and a cysteine conjugated antibody-drug conjugate (ADCs), namely brentuximab-vedotin, could denaturate. For this purpose, some experiments were carried out in HIC conditions using various organic modifier in natures and proportions, different mobile phase temperatures and also different pHs. Indeed, improper analytical conditions in hydrophobic interaction chromatography (HIC) may create reversed-phase (RP) like harsh conditions and therefore protein denaturation.

Pushing quantitation limits in micro UHPLC-MS/MS analysis of steroid hormones by sample dilution using high volume injection.

Ultratrace analysis of sample components requires excellent analytical performance in terms of limits of quantitation (LoQ). Micro UHPLC coupling with sensitive tandem mass spectrometry provides state of the art solutions for such analytical problems. Decreased column volume in micro LC limits the injectable sample volume.

Challenges in liquid chromatographic characterization of proteins.

Various liquid chromatographic techniques are considered standard analytical methods in proteins characterization. These methods provide essential information for drug approval, for biological and life sciences. On the other hand, there are some issues and challenges which have to be taken into account when analyzing these biopharmaceuticals.

Systematic evaluation of mobile phase additives for the LC-MS characterization of therapeutic proteins.

Trifluoroacetic acid (TFA) is commonly used as mobile phase additive for the analysis of proteins in reversed phase liquid chromatography (RPLC). Due to its interesting features, it provides symmetrical and narrow peak shapes for proteins, but decreases mass spectrometric sensitivity through ion-pairing and spray destabilizing. Since RPLC-MS is an important technique for the characterization of proteins, some alternative MS-compatible mobile phases may be required.

Recovery of Proteins Affected by Mobile Phase Trifluoroacetic Acid Concentration in Reversed-Phase Chromatography.

It was found that recoveries of proteins depend on trifluoroacetic acid concentration in the mobile phase and showed maximum in the range of 0.01-0.1 v/v%.

Evaluation of new superficially porous particles with carbon core and nanodiamond-polymer shell for proteins characterization.

A new superficially porous material possessing a carbon core and nanodiamond-polymer shell and pore size of 180Å was evaluated for the analysis of large proteins. Because the stationary phase on this new support contains a certain amount of protonated amino groups within the shell structure, the resulting retention mechanism is most probably a mix between reversed phase and anion exchange. However, under the applied conditions (0.

HPLC enrichment/isolation of proteins for post-translational modification studies from complex mixtures.

The paper describes a macroporous RP-HPLC method for separation and isolation/enrichment of proteins from complex mixtures. The method is robust and efficient; using 2.1 or 4.

Influence of acid-induced conformational variability on protein separation in reversed phase high performance liquid chromatography.

Influence of acid concentration in the mobile phase on protein separation was studied in a wide concentration range using trifluoroacetic acid (TFA) and formic acid (FA). At low, 0.001-0.