A thorough evaluation of the quality, reproducibility, and variability of bottom-up proteomics data is necessary at every stage of a workflow from planning to analysis. We share real-world case studies applying adaptable quality control (QC) measures to assess sample preparation, system function, and quantitative analysis. System suitability samples are repeatedly measured longitudinally with targeted methods, and we share examples where they are used on three instrument platforms to identify severe system failures and track function over months to years. Internal QCs incorporated at protein and peptide-level allow our team to assess sample preparation issues and to differentiate system failures from sample-specific issues. External QC samples prepared alongside our experimental samples are used to verify the consistency and quantitative potential of our results during batch correction and normalization before assessing biological phenotypes. We combine these controls with rapid analysis using Skyline, longitudinal QC metrics using AutoQC, and server-based data deposition using PanoramaWeb. We propose that this integrated approach to QC be used as a starting point for groups to facilitate rapid quality control assessment to ensure that valuable instrument time is used to collect the best quality data possible.

Size exclusion chromatography (SEC) is a foundational analytical method to assess product purity of biological molecules. To ensure accurate and reproducible data that meet regulatory agency standards, it is critical to monitor the chromatographic column with efficient and continuous approaches. In this study, 19 SEC columns (Waters Acquity BEH200) were evaluated using an in-house monoclonal antibody made at Regeneron. System suitability parameters (SSPs) were used to monitor the performance of the SEC assay, including USP resolution, USP plate count, USP tailing factor, asymmetry factor, elution time, peak width, and peak height. A general linear model was built and revealed that elution time, peak width, asymmetry factor, and tailing factor increased with injection number, while peak height, resolution, and plate count decreased. After 1000 injections, tailing factor and peak width increased by more than 10%, while resolution and plate count decreased by more than 10% from their respective starting values.

An amino acid sequence variant (SV) is defined as an unintended amino acid substitution in protein drug products. SVs contribute to product heterogeneity and can potentially impact product quality, safety, immunogenicity, and efficacy. The analysis of biotherapeutics for SVs is important throughout the product life cycle including clone selection, development of nutrient feed strategies, commercial manufacturing process, and post-approval changes to monitor product quality. The proposed analytical procedure for SVs consists of both qualitative (identification of SVs) and quantitative (quantitation of identified SVs) components. The complexities of SV analysis and the variety of current procedures highlight the need for a systematic approach for assessing the capability of these methodologies to reliably identify and quantitate SVs in biotherapeutics. We described here a \"spike-control\" approach for evaluating SV analytical procedure. The concept was adopted from quality control samples routinely used in analytical procedure validation. One FDA approved monoclonal antibody (mAb) was spiked with accurate amounts of highly homologous mAb to create mAb samples containing low yet accurate levels of \"artificial\" SVs. Spike-control samples were denatured, reduced, alkylated, digested and then analyzed by high resolution Orbitrap mass spectrometry. In silico analysis revealed four single amino acid differences between the two mAbs that could be used to represent SVs in the spike-control samples. All four \"artificial\" SVs were reliably identified by the current workflow. Analytical range (0.01% to 2%), accuracy and precision of identified SVs have also been evaluated. Overall, spike-control sample(s) helped to demonstrate that the SV analytical procedure (i.e., sample preparation, LC separation, mass spectrometry determinations and bioinformatic software) was fit for purpose and suitable for the identification and quantitation of SVs at a pre-determined threshold.

The control of somatropin products according to the monographs of the European Pharmacopoeia (Ph. Eur.) requires a system suitability preparation for the test for related proteins by liquid chromatography. A preparation consisting in a mixture of somatropin and desamidosomatropin, such as the Ph. Eur. Somatropin/desamidosomatropin resolution mixture Chemical Reference Substance (CRS), is to be used to ascertain adequate resolution of the chromatographic setup. Due to low stocks, the Biological Standardisation Programme (BSP) of the Council of Europe and the European Union ran a study to establish a new batch of this system suitability CRS. A freeze-dried candidate batch (cCRS2) was produced and tested at the European Directorate for the Quality of Medicines and HealthCare (EDQM, Council of Europe). The resolution between the peaks due to somatropin and desamidosomatropin was 1.7 and the symmetry factor for the somatropin peak was 1.2. The mean percentage area of the desamidosomatropin peak was 14.6 %. These results showed that cCRS2 is suitable for its intended purpose. Based on these data, in May 2020 the Ph. Eur. Commission established the candidate batch as Ph. Eur. Soma-tropin/desamidosomatropin resolution mixture CRS batch 2.

It is critical to the success of any chromatography-based assay that the performance of the LC instrument be checked for its readiness and ability to perform the intended analysis. This includes gaging the suitability of a system to fulfill the purpose of different types of methods. One type of analysis that requires special consideration is the analysis of compounds which are prone to a particular form of non-specific binding, namely metal adsorption, where analytes interact and potentially adsorb to metal contained within the chromatographic flow path. For an analysis of compounds which are susceptible to metal adsorption, ideally a system suitability test would be performed to ensure there will not be any sample loss or detrimental peak shape effects resulting from potential analyte-to-metal interactions. To help chromatographers assess system inertness concerns like this, we have developed a method of testing LC systems for metal interactions using adenosine 5\'-(α,β-methylene)diphosphate (AMPcP). This nucleotide analog has been confirmed to have a propensity to adsorb to titanium and stainless-steel frits and is resistant to hydrolysis and stable to long-term storage and repeat use (as is befitting of any reagent proposed for system suitability testing). AMPcP has been used in a flow injection test (no column in-line) to monitor for losses in recovery and peak shape perturbations that can potentially be present in any chromatography system manufactured with one or more metal based components. In this approach, sequential injections of AMPcP were made without a column and various peak attributes were monitored and ultimately correlated to the amount of metal surface area in the flow path. The ability of this method to discriminate between inert chromatographic surfaces versus exposed metal surfaces was verified by comparing peak areas, peak shapes, and injection repeatability for AMPcP using a UHPLC equipped with MP35N metal alloy components versus an equivalent UHPLC equipped with an ethylene bridged hybrid organic-inorganic surface (or so-called hybrid surface technology). Injections of caffeine were also explored to establish a negative control for this system suitability measurement. Caffeine does not interact with metal surfaces and can therefore give an instrument specific representation of peak shape and dispersion as well as an indication of overall mechanical system performance. Additionally, replicate injections of AMPcP and caffeine onto a UHPLC partially configured with hybrid surface technology (HST) readily identified exposed metal surfaces through an increased peak area relative standard deviation as well as a reduction in absolute recovery. Finally, a novel visualization tool was developed to provide an alternative method of determining system inertness without having to perform chromatographic calculations but instead a graphical peak shape comparison between a negative control, caffeine, and the metal sensitive AMPcP test probe.

Antibody drug conjugates (ADCs) represent a rapidly growing modality for the treatment of numerous oncology indications. The complexity of analytical characterization method development is increased due to the potential for synthetic intermediates and process-related impurities. In addition, the cytotoxicity of such materials provides an additional challenge with regard to handling products and/or sharing materials with analytical collaborators and/or vendors for technology development. Herein, we have utilized a site-specific chemoenzymatic glycoconjugation strategy for preparing ADC mimetics composed of the NIST monoclonal antibody (NISTmAb) conjugated to non-cytotoxic payloads representing both small molecules and peptides. The materials were exhaustively characterized with high-resolution mass spectrometry-based approaches to demonstrate the utility of each analytical method for confirming the conjugation fidelity as well as deep characterization of low-abundance synthetic intermediates and impurities arising from payload raw material heterogeneity. These materials therefore represent a widely available test metric to develop novel ADC analytical methods as well as a platform to discuss best practices for extensive characterization.

UNASSIGNED: The aim of the study was to develop a simple, highthroughput and sensitive LC-MS/MS method and apply to a bioequivalence study of montelukast, a light sensitive drug.
UNASSIGNED: The effects of organic modifiers in mobile phase, protein precipitation agent to plasma sample ratio, and light on montelukast stability in unprocessed and processed human plasma, were evaluated. Validation was conducted in accordance with European Medicines Agency Guideline on bioanalytical method validation.
UNASSIGNED: No interference peak was observed when acetonitrile was used as an organic modifier. Acetonitrile to plasma ratio of 4:1 produced clean plasma sample. Approximately 3 % of cis isomer was detected in unprocessed plasma samples while 21 % of cis isomer was detected in processed plasma samples after exposing to fluorescent light for 24h. The standard calibration curve was linear over 3.00-1200.00 ng/mL. All method validation parameters were within the acceptance criteria.
UNASSIGNED: The validated method was successfully applied to a bioequivalence study of two montelukast formulations involving 24 healthy Malaysian volunteers. The light stability of a light sensitive drug in unprocessed and processed human plasma samples should be studied prior to pharmacokinetic/bioequivalence studies. Measures could then be taken to protect the analyte in human plasma from light degradation.

Preprocessing data in a reproducible and robust way is one of the current challenges in untargeted metabolomics workflows. Data curation in liquid chromatography-mass spectrometry (LC-MS) involves the removal of biologically non-relevant features (retention time, m/z pairs) to retain only high-quality data for subsequent analysis and interpretation. The present work introduces TidyMS, a package for the Python programming language for preprocessing LC-MS data for quality control (QC) procedures in untargeted metabolomics workflows. It is a versatile strategy that can be customized or fit for purpose according to the specific metabolomics application. It allows performing quality control procedures to ensure accuracy and reliability in LC-MS measurements, and it allows preprocessing metabolomics data to obtain cleaned matrices for subsequent statistical analysis. The capabilities of the package are shown with pipelines for an LC-MS system suitability check, system conditioning, signal drift evaluation, and data curation. These applications were implemented to preprocess data corresponding to a new suite of candidate plasma reference materials developed by the National Institute of Standards and Technology (NIST; hypertriglyceridemic, diabetic, and African-American plasma pools) to be used in untargeted metabolomics studies in addition to NIST SRM 1950 Metabolites in Frozen Human Plasma. The package offers a rapid and reproducible workflow that can be used in an automated or semi-automated fashion, and it is an open and free tool available to all users.

The separation of enantiomers is an important requirement during the entire drug life cycle in the pharmaceutical industry. High-performance liquid chromatography and supercritical fluid chromatography (SFC) are the main chromatographic techniques used to separate enantiomers. Since chiral stationary phases are often extensively used once a method has been developed, columns will age and must be replaced after a certain period. However, no practical guidelines exist to determine when a column is deteriorated or to decide whether a transfer to another column (with the same chiral selector) is successful. In this study, a system suitability limit for resolution was defined, based on an intermediate (time-different) precision study in SFC on four immobilized polysaccharide-based columns that only differed in manufacturer or particle size. This system suitability limit could be used to decide on column deterioration or as a requirement to evaluate whether a separation transfer was successful. Some method adaptations may be necessary to obtain successful transfers. An approach was proposed, which helped the analyst to make successful transfers. Graphical abstract.

Substances leached from pharmaceutical manufacturing systems, packages, and/or medical devices can be administered to a patient during a clinical therapy and can adversely affect the therapy and/or patient safety. Thus, extracts or drug products are chromatographically screened to discover, identify, and quantify these unspecified foreign impurities. Although screening methods have achieved a high degree of technical and practical sophistication, they are not without issues in terms of accomplishing these three functions. In this third (and last) part of the series, errors of implementation are addressed. An error of implementation occurs when a capable method and/or a proper data processing procedure is implemented in such a way that the intrinsic capabilities of either the method or the processing procedure (or both) are compromised. System suitability assessment establishes that a method, as executed at the time of use, has been properly set up and implemented, thus revealing errors of implementation. System suitability data, captured in a database, can be processed to establish trends in system performance, where trends in declining performance can establish a system\'s useful operating lifetime, serve as an early warning of imminent system failure, and/or act as a trigger for system maintenance. Additionally, this manuscript considers how the existence, size, and use of a chromatographic database provides a measure of a testing laboratory\'s level of \"good science\". Lastly, the manuscript considers the database as an enabler of advances in information management and impact assessment.