Optimizing sample preparation and nLC-MS/MS for mAB characterization

An Improved Procedure for Enzymatic Digestion of Polyvinylidene Difluoride-Bound Proteins for Internal Sequence Analysis

Development and characterisation of a method for determining N-terminal heterogeneity in the human recombinant follitropin β-subunit.

Nevirapine (NVP), used for the treatment of HIV/AIDS, exhibits unpredictable oral bioavailability, has a poor side effect profile and requires frequent dosing. Niosomes are novel drug delivery systems that have the potential to overcome these challenges. A thin layer hydration approach was used to produce niosomes and optimisation was undertaken using design of experiments (DoE) and response surface methodology (RSM) establish and identify parameters that may affect the manufacture of niosomes. The impact of cholesterol and surfactant content, hydration time and temperature on manufacture was investigated. Critical quality attributes (CQA) in respect of particle size (PS), entrapment efficiency (EE), polydispersity index (PDI) and the amount of NVP released at 48 hours was also assessed. The optimised niosome composition was identified and manufactured and the CQA characterised prior to placing the batch on stability for 12 weeks at 4±2 °C and 22±2 °C. The PS, PDI, EE and % NVP released at 48 h was 523.36±23.16 nm, 0.386±0.054, 96.8 % and 25.3 % for niosomes manufactured with Span® 20. Similarly, the parameters were 502.87±21.77 nm and 0.394±0.027, 98.0 % and 25.0 % for mean PS, PDI, EE and %NVP released at 48 h for Span® 80 niosomes. All characterisation was undertaken on the day of manufacture. In conclusion, a simple, cheap, rapid and precise method of manufacture of NVP niosomes was developed, validated and optimised using DoE and RSM and the product exhibited the target CQA.

Peptide mapping with mass spectrometry (MS) is an important tool for protein characterization in the biopharmaceutical industry. Historically, peptide mapping monitors post-translational modifications (PTMs) of protein products and process intermediates during development. Multi-attribute monitoring (MAM) methods have been used previously in commercial release and stability testing panels to ensure control of selected critical quality attributes (CQAs). Our goal is to use MAM methods as part of an overall analytical testing strategy specifically focused on CQAs, while removing or replacing historical separation methods that do not effectively distinguish CQAs from non-CQAs due to co-elution. For example, in this study, we developed a strategy to replace a profile-based ion-exchange chromatography (IEC) method using a MAM method in combination with traditional purity methods to ensure control of charge variant CQAs for a commercial antibody (mAb) drug product (DP). To support this change in commercial testing strategy, the charge variant CQAs were identified and characterized during development by high-resolution LC-MS and LC-MS/MS. The charge variant CQAs included PTMs, high molecular weight species, and low molecular weight species. Thus, removal of the IEC method from the DP specification was achieved using a validated LC-MS MAM method on a QDa system to directly measure the charge variant PTM CQAs in combination with size exclusion chromatography (SE-HPLC) and capillary electrophoresis (CE-SDS) to measure the non-PTM charge variant CQAs. Bridging data between the MAM, IEC, and SE-HPLC methods were included in the commercial marketing application to justify removing IEC from the DP specification. We have also used this MAM method as a test for identity to reduce the number of QC assays. This strategy has received approvals from several health authorities.

Scale-up approaches for film coating process have been established for each type of film coating equipment from thermodynamic and mechanical analyses for several decades. The objective of the present study was to establish a versatile scale-up approach for film coating process applicable to commercial production that is based on critical quality attribute (CQA) using the Quality by Design (QbD) approach and is independent of the equipment used. Experiments on a pilot scale using the Design of Experiment (DoE) approach were performed to find a suitable CQA from surface roughness, contact angle, color difference, and coating film properties by terahertz spectroscopy. Surface roughness was determined to be a suitable CQA from a quantitative appearance evaluation. When surface roughness was fixed as the CQA, the water content of the film-coated tablets was determined to be the critical material attribute (CMA), a parameter that does not depend on scale or equipment. Finally, to verify the scale-up approach determined from the pilot scale, experiments on a commercial scale were performed. The good correlation between the surface roughness (CQA) and the water content (CMA) identified at the pilot scale was also retained at the commercial scale, indicating that our proposed method should be useful as a scale-up approach for film coating process.

In the peptide mapping reduction process for monoclonal antibodies (mAbs) and other proteins, the conventional reducing reagents β-mercaptoethanol (β-ME) and dithiothreitol (DTT) pose challenges due to their strong odor and toxicity at high concentrations. Cysteine (Cys), an essential amino acid for new protein synthesis, is an overlooked, nontoxic, and odorless reducing agent. This study presents a novel peptide mapping method using Cys as the reducing agent. We explored the reducing effect of Cys at different concentrations and pH levels for peptide mapping analysis of a specific mAb (mAb-1), using DTT as a positive control. RP-HPLC analysis, including UV chromatogram comparison and overall similarity calculation, was conducted for comparison. LC-MS analysis was subsequently utilized to characterize the primary sequence of mAb-1. We also applied the method to other mAbs or proteins to demonstrate its wide applicability. The UV chromatogram and overall similarity of Cys as a reducing agent at concentrations ranging from 10 to 40mM and pH levels between 7.0 and 11.0 were consistent with those of the positive control. Reduced concentrations of Cys or lower pH levels compromised reducing efficacy. This novel reducing method proficiently characterized the primary sequence of mAb-1, achieving an overall sequence coverage of 97%. In the analysis of other mAbs or proteins, the peptide mapping results also showed high consistency. Cys exhibits a reducing ability comparable to DTT and possesses the advantageous characteristics of being nontoxic and odorless, making it a potential alternative for disulfide bond reduction and peptide mapping analysis of proteins and mAbs.

Optimized 2DE sample preparation protocols that maximize total protein recovery are fundamental to improving proteome capture and increasing the utility of 2DE, which is in part limited by inadequate recovery of proteins with diverse physicochemical properties. Maintaining protein solubility is an important factor for protein recovery, but the multitude of solubility-enhancing agents and the relatively low-throughput nature of 2DE limit the systematic study of sample preparation. In this work, design of experiment (DOE) approaches are used to optimize protein recovery by altering the levels of four solubility-enhancing agents (urea, DTT, CHAPS, and SDS) in the initial suspension solution. Protein recovery is quantified by a total protein concentration assay, which is demonstrated to be representative of SDS-PAGE and 2DE recovery. DOE methodologies are presented as relatively high-throughput procedures for optimizing 2DE sample preparation parameters for a variety of sample types. Optimal suspension solution compositions are shown to vary across a model protein solution (no urea or DTT), Chinese hamster ovary (CHO) cell lysate (8 M urea, ≥2% CHAPS, ≥32.5 mM DTT), and Escherichia coli cell lysate (8 M urea, 4% CHAPS, 65 mM DTT), with optimized conditions increasing 2DE protein recovery at least 50% compared to suboptimal conditions.

Addendum: Digestion and depletion of abundant proteins improves proteomic coverage.

Analysis of monoclonal antibody product heterogeneity resulting from alternate cleavage sites of signal peptide

Rapid high-throughput antibody analysis using microwave-assisted digestion.

Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding <0.1 cent in material costs to the price of each tip. The system allows fast loading with low backpressure (>300 micro/min for the packed column using manual force) while eliminating the possibility of blocking. The loading capacity of C18-StageTips (column bed: 0.4 mm diameter, 0.5 mm length) is 2-4 microg of protein digest, which can be increased by using larger diameter or stacked disks. Five femtomole of tryptic BSA digest could be recovered quantitatively. We have found that the Stage system is well-suited as a universal sample preparation system for proteomics.

The implementation of 'Analytical Quality by Design' (AQbD) is currently recognised as a methodical and scientific approach to liquid chromatographic (LC) method development. It begins with defining the Analytical Target Profile (ATP), identifying Critical Method Parameters (CMPs) and Critical Quality Attributes (CQAs) or responses. This process also includes risk assessment study by Design of Experiment (DoE) and to optimise LC effectively. The next step is to create an analytical design space and implement a control strategy that will allow for continuous method improvement over the life of the method. Understanding the AQbD principles, methodologies, techniques and applications towards the high-performance liquid chromatography (HPLC) method lifecycle is considered as essential in today's pharmaceutical landscape. As industry demands for quality assurance and regulatory compliance, this review paper briefly discusses the AQbD principles and methodology in HPLC method lifecycle. Also, the paper covers AQbD components such as identification of ATP, CQA, MODR (method operable design region), control strategies and continuous method monitoring. In addition, it also covers regulatory perspectives on AQbD, obstacles and potential outcomes in the development of pharmaceutical analytical methods. These aspects provide valuable insights into the application of AQbD in the field of LC.

A quality-by-design approach was adopted to develop telmisartan potassium (TP) tablets, which were bioequivalent with the commercially available Micardis® (telmisartan free base) tablets. The dissolution pattern and impurity profile of TP tablets differed from those of Micardis® tablets because telmisartan free base is poorly soluble in water. After identifying the quality target product profile and critical quality attributes (CQAs), drug dissolution, and impurities were predicted to be risky CQAs. To determine the exact range and cause of risks, we used the risk assessment (RA) tools, preliminary hazard analysis and failure mode and effect analysis to determine the parameters affecting drug dissolution, impurities, and formulation. The range of the design space was optimized using the face-centered central composite design among the design of experiment (DOE) methods. The binder, disintegrant, and kneading time in the wet granulation were identified as X values affecting Y values (disintegration, hardness, friability, dissolution, and impurities). After determining the design space with the desired Y values, the TP tablets were formulated and their dissolution pattern was compared with that of the reference tablet. The selected TP tablet formulated using design space showed a similar dissolution to that of Micardis® tablets at pH 7.5. The QbD approach TP tablet was bioequivalent to Micardis® tablets in beagle dogs.

Integrated Application of Quality-by-Design Principles to Drug Product Development: A Case Study of Brivanib Alaninate Film–Coated Tablets

Objective: The current probing aims to investigate the Artificial Intelligence (AI) powered Design of Experiments (DoE) to optimize Abiraterone acetate loaded Gelatin Nanoparticles (AGNPs) with the desired Critical Quality Attributes (CQA) for increasing oral bioavailability of abiraterone acetate. Methods: AGNPs were formulated using the desolvation method, guided by a quality-by-design (QbD) approach, to identify CQA. The proposed DoE was a Central Composite Design (CCD) that was done to determine the influence of gelatin, tween 80, and genipin on particle size and Drug Entrapment Efficiency (DEE). Further optimization was performed with Artificial Neural Networks (ANN) to refine the predictive models. Results: The ANN model was developed using data from the CCD. K-fold cross-validation was implemented for training and validating the model. Both training and validation sets' R-squared values were closer to 1, confirming that the algorithms correctly characterized the predictive models. Characterization studies confirmed that optimized AGNPs had particle size (123.4±0.06 nm), poly dispersibility index (0.047±0.07), DEE (88.23±0.26 %), zeta potential (+35.78±0.24 mV), and controlled drug release (96.32±0.78 % over 12 h). Conclusion: The results show that nanoparticles developed according to the criteria set by CQA to enhance oral bioavailability, and an AI-integrated CCD approach optimally worked for AGNPs, which can deliver abiraterone acetate orally. The infusion of advanced technologies ANN undoubtedly holds excellent potential for exciting discoveries in nanomedicine and enables future innovations.