Monitoring stability indicating impurities and aldehyde content in lipid nanoparticle raw material and formulated drugs.

Lipid nanoparticles (LNPs) are designed to protect and transport sensitive payloads or active pharmaceutical ingredients as part of new therapeutic modalities. As a multi-component particle, a high degree of quality control is necessary to ensure raw materials are free of critical impurities that could adversely impact the drug product. In this study, we demonstrate a reversed phase liquid chromatography method hyphenated with a single quadrupole mass spectrometer (RPLC-MS) as an alternative platform to methods that incorporate evaporative light scattering or charged aerosol detectors in the detection and quantitation of critical impurities associated with LNPs. The proposed RPLC-MS method offers an increase of up to 2 orders of magnitude in dynamic range and 3 orders of magnitude in sensitivity in the analysis of impurities associated with LNPs compared to conventional detectors. Access to complementary mass data enabled the detection and identification of stability indicating impurities as part of stress studies carried out on an ionizable lipid. In addition to confirmation of peak identity, complementary mass data was also used to assess residual aldehydes in raw material and formulated LNPs in accordance with regulatory guidance. Following derivatization using 2,4-dinitrophenylhydrazine, aldehyde content in the ionizable lipid raw material was determined to exceed the reporting threshold of 0.05% in 30% of the test cases. The experimental findings observed in this study demonstrate the utility of the proposed RPLC-MS method in the identification and monitoring of stability-indicating attributes associated with LNPs as part of current Good Manufacturing Practices for improved consumer safety in drug products.

Reducing metal-ion mediated adsorption of acidic peptides in RPLC-based assays using hybrid silica chromatographic surfaces.

In this study we evaluate column hardware exhibiting a novel hybrid silica surface in its ability to mitigate metal-induced adsorption artifacts such as chromatographic peak tailing for acidic amino acid residue containing peptides. Using a conventional reversed-phase liquid chromatography (RPLC)-based method, chromatographic performance of a peptide map was compared using a traditional stainless-steel column and an equivalent column bearing a novel hybrid silica surface. Tailing factors for six peptides containing acidic amino acid residues (Tf ≥ 1.50) were observed to be reduced up to 80% to a nominal value Tf ≤ 1.2 with R.S.D. % ≤ 4%. Furthermore, recovery for two of the identified peptides exhibited increased recovery in addition to reduced peak tailing when using the column bearing the hybrid silica surface. Performance was unaffected for peaks where there were no implications of metal induced effects. Collectively, this study demonstrates that the novel hybrid silica surface can effectively reduce peak tailing for acidic residue containing peptides.

High-Throughput Analysis of Fluorescently Labeled N-Glycans Derived from Biotherapeutics Using an Automated LC-MS-Based Solution.

Protein glycosylation can impact the efficacy and safety of biotherapeutics and therefore needs to be well characterized and monitored throughout the drug product life cycle. Glycosylation is commonly assessed by fluorescent labeling of released glycans, which provides comprehensive information of the glycoprofile but can be resource-intensive regarding sample preparation, data acquisition, and data analysis. In this work, we evaluate a comprehensive solution from sample preparation to data reporting using a liquid chromatography-mass spectrometry (LC-MS)-based analytical platform for increased productivity in released glycan analysis. To minimize user intervention and improve assay robustness, a robotic liquid handling platform was used to automate the release and labeling of N-glycans within 2 h. To further increase the throughput, a 5 min method was developed on a liquid chromatography-fluorescence-mass spectrometry (LC-FLR-MS) system using an integrated glycan library based on retention time and accurate mass. The optimized method was then applied to 48 released glycan samples derived from six batches of infliximab to mimic comparability testing encountered in the development of biopharmaceuticals. Consistent relative abundance of critical species such as high mannose and sialylated glycans was obtained for samples within the same batch (mean percent relative standard deviation [RSD] = 5.3%) with data being acquired, processed, and reported in an automated manner. The data acquisition and analysis of the 48 samples were completed within 6 h, which represents a 90% improvement in throughput compared with conventional LC-FLR-based methods. Together, this workflow facilitates the rapid screening of glycans, which can be deployed at various stages of drug development such as process optimization, bioreactor monitoring, and clone selections, where high-throughput and improved productivity are particularly desired.

Application of mobile phase additives to reduce metal-ion mediated adsorption of non-phosphorylated peptides in RPLC/MS-based assays.

Metal-ion mediated adsorption in liquid chromatography has been identified as a contributing factor in poor peak shape, tailing, and diminished recovery of compounds prone to cation exchange-like interaction with metal-based activity sites. Peptides that exhibit negative charge-bearing amino acids such as aspartic acid and glutamic acid are particularly sensitive to metal-ion mediated adsorption in RPLC/MS-based separations when using weak acids (e.g. formic acid) as mobile phase additives. Citric acid and medronic acid as metal complexing mobile phase additives were evaluated for their ability to mitigate metal-ion mediated adsorption in RPLC/MS-based peptide mapping assays. In this study, chromatographic performance was stabilized with peak tailing for peptides of interest reduced by as much as 40% in the presence of a chelator at a mobile phase concentration of 1 ppm. Performance gains were observed to be stable over a 67-hour time study with an average USP tailing factor of 1.00, % RSD = 0.64. The stabilizing effect of the chelator improved peptide mapping assay robustness with relative peak areas for target impurities calculated at 2.28% (% R.S.D. = 2.36) and 2.40% (% R.S.D. = 2.37). Collectively this study demonstrates that chelators as mobile phase additives offers a means to improve chromatographic performance for biomolecules sensitive to metal-ion mediated adsorption under formic acid-based RPLC conditions.

A sensitive multidimensional method for the detection, characterization, and quantification of trace free drug species in antibody-drug conjugate samples using mass spectral detection.

Conjugation processes and stability studies associated with the production and shelf life of antibody-drug conjugates (ADCs) can result in free (non-conjugated) drug species. These free drug species can increase the risk to patients and reduce the efficacy of the ADC. Despite stringent purification steps, trace levels of free drug species may be present in formulated ADCs, reducing the therapeutic window. The reduction of sample preparation steps through the incorporation of multidimensional techniques has afforded analysts more efficient methods to assess trace drug species. Multidimensional methods coupling size-exclusion and reversed phase liquid chromatography with ultra-violet detection (SEC-RPLC/UV) have been reported, but offer limited sensitivity and can limit method optimization. The current study addresses these challenges with a multidimensional method that is specific, sensitive, and enables method control in both dimensions via coupling of an on-line solid phase extraction column to RPLC with mass spectral detection (SPE-RPLC/MS). The proposed method was evaluated using an antibody-fluorophore conjugate (AFC) as an ADC surrogate to brentuximab vedotin and its associated parent maleimide-val-cit-DSEA payload and the derived N-acetylcysteine adduct formed during the conjugation process. Assay sensitivity was found to be 2 orders more sensitive using MS detection in comparison to UV-based detection with a nominal limit of quantitation of 0.30 ng/mL (1.5 pg on-column). Free-drug species were present in an unadulterated ADC surrogate sample at concentrations below 7 ng/mL, levels not detectable by UV alone. The proposed SPE-RPLC/MS method provides a high degree of specificity and sensitivity in the assessment of trace free drug species and offers improved control over each dimension, enabling straightforward integration into existing or novel workflows.

Reduction of metal adducts in oligonucleotide mass spectra in ion-pair reversed-phase chromatography/mass spectrometry analysis.

RATIONALE: Electrospray ionization mass spectrometry (ESI-MS)-based techniques commonly used in oligonucleotide analyses are known to be sensitive to alkali metal adduct formation. Adducts directly impact the sensitivity of MS-based analyses as the available charge is distributed across the parent peak and adduct(s). The current study systematically evaluated common liquid chromatography (LC) components in LC/ESI-MS configurations used in oligonucleotide analysis to identify metal adduct contributions from LC instrumentation. METHODS: A UPLC liquid chromatography system was configured with a single quadrupole MS detector (ACQUITY QDa, Waters Corp.) to monitor adduct formation in oligonucleotide separations. An ion-pairing mobile phase comprised of 15 mM triethylamine and 400 mM hexafluoro-2-propanol was used in conjunction with an oligonucleotide separation column (Waters OST BEH C18, 2.1 mm × 50 mm) for all separations. A 10-min method was used to provide statistical figures of merit and evaluate adduct formation over time. RESULTS: Trace alkali metal salts in the mobile phase and reagents were determined to be the main source of metal salt adducts in LC/ESI-MS-based configurations. Non-specific adsorption sites located throughout the fluidic path contribute to adduct formation in oligonucleotide analyses. Ion-pairing mobile phases prepared at neutral or slightly basic pH result in up to a 57% loss of spectral abundance to adduct formation in the current study. CONCLUSIONS: Implementation of a short low pH reconditioning step was observed to effectively displace trace metal salts non-specifically adsorbed to surfaces in the fluidic path and was able to maintain an average MS spectral abundance ≥94% with a high degree of repeatability (relative standard deviation (R.S.D.) 0.8%) over an extended time study. The proposed method offers the ability to rapidly regenerate adsorption sites with minimal impact on productivity while retaining assay sensitivity afforded by MS detection with reduced adduct formation. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.

A rapid on-line method for mass spectrometric confirmation of a cysteine-conjugated antibody-drug-conjugate structure using multidimensional chromatography.

Cysteine-conjugated antibody-drug conjugates (ADCs) are manufactured using controlled partial reduction and conjugation chemistry with drug payloads that typically occur in intervals of 0, 2, 4, 6, and 8. Control of heterogeneity is of particular importance to the quality of ADC product because drug loading and distribution can affect the safety and efficacy of the ADC. Liquid chromatography ultra-violet (LC-UV)-based methods can be used to acquire the drug distribution profiles of cysteine-conjugated ADCs when analyzed using hydrophobic interaction chromatography (HIC). However, alternative analysis techniques are often required for structural identification when conjugated drugs do not possess discrete ultra-violet absorbance properties for precise assessment of the drug-to-antibody ratio (DAR). In this study, multidimensional chromatography was used as an efficient method for combining non-compatible techniques, such as HIC, with analysis by mass spectrometry (LC/LC/QTOF-MS) for rapid on-line structural elucidation of species observed in HIC distribution profiles of cysteine-conjugated ADCs. The methodology was tested using an IgG1 mAb modified by cysteine conjugation with a non-toxic drug mimic. Structural elucidation of peaks observed in the HIC analysis (1(st) dimension) were successfully identified based on their unique sub-unit masses via mass spectrometry techniques once dissociation occurred under denaturing reversed phase conditions (2(nd) dimension). Upon identification, the DAR values were determined to be 2.83, 4.44, and 5.97 for 3 drug load levels (low-, medium-, and high-loaded ADC batches), respectively, based on relative abundance from the LC-UV data. This work demonstrates that multidimensional chromatography coupled with MS, provides an efficient approach for on-line biotherapeutic characterization to ensure ADC product quality.

RPLC of intact proteins using sub-0.5 µm particles and commercial instrumentation.

This paper addresses whether one can gain an improvement in speed or resolution with a silica colloidal crystal (SCC) of nonporous 470 nm particles when using a commercial nano-UHPLC. Compared to a capillary packed with nonporous 1.3 µm particles and the same C4 bonded phase, the peak width for BSA is decreased by a factor of 6.8 for the SCC. Some of this improvement is attributable to slip flow since the ratio of particle diameters is only 2.8. Resolution in protein separations was compared for a 2-cm capillary of SCC vs a 5-cm column of porous 1.7 µm particles. Both used a C4 bonded phase, and on-column fluorescence detection was used for the SCC. Split flow (5:1) before the SCC decreased the gradient delay time to 0.4 min and the injected volume to 0.4 nL. For variants from the labeling of BSA, the SCC had a 5-fold higher speed and 2-fold higher resolution than did the commercial column. For a monoclonal antibody and its aggregates, the SCC had a 3-fold higher speed and a 3-fold higher resolution compared to the commercial column. The SCC gave baseline resolution of the monomer, dimer and trimer in 5 min. The results show that a significant advantage can be gained using a commercial instrument with the SCC, despite the instrument not being designed for use with such small particles.

Silica colloidal crystals as emerging materials for high-throughput protein electrophoresis.

Silica colloidal crystals are a new type of media for protein electrophoresis, and they are assessed for their promise in rapidly measuring aggregation of monoclonal antibodies. The nature of silica colloidal crystals is described in the context of the need for a high-throughput separation tool for optimizing the formulations of protein drugs for minimal aggregation. The fundamental relations between molecular weight and mobility in electrophoresis are used to make a theoretical comparison of selectivity between gels and colloidal crystals. The results show that the selectivity is similar for these media, but slightly higher, 10%, for gels, and the velocity is inherently lower than that for gels due to the smaller free volume fraction. These factors are more than compensated for by lower broadening in colloidal crystals. These new media give plate heights of only 0.15 µm for the antibody monomer and 0.42 µm for the antibody dimer. The monoclonal antibody is separated from its dimer in 72 s over a distance of only 6.5 mm. This is five times faster than size-exclusion chromatography, with more than tenfold miniaturization, and amenable to parallel separations, all of which are promising for the design of high-throughput devices for optimizing protein drug formulations.

Modeling of protein electrophoresis in silica colloidal crystals having brush layers of polyacrylamide.

Sieving of proteins in silica colloidal crystals of millimeter dimensions is characterized for particle diameters of nominally 350 and 500 nm, where the colloidal crystals are chemically modified with a brush layer of polyacrylamide. A model is developed that relates the reduced electrophoretic mobility to the experimentally measurable porosity. The model fits the data with no adjustable parameters for the case of silica colloidal crystals packed in capillaries, for which independent measurements of the pore radii were made from flow data. The model also fits the data for electrophoresis in a highly ordered colloidal crystal formed in a channel, where the unknown pore radius was used as a fitting parameter. Plate heights as small as 0.4 µm point to the potential for miniaturized separations. Band broadening increases as the pore radius approaches the protein radius, indicating that the main contribution to broadening is the spatial heterogeneity of the pore radius. The results quantitatively support the notion that sieving occurs for proteins in silica colloidal crystals, and facilitate design of new separations that would benefit from miniaturization.

Effects of lead and mercury on the blood proteome of children.

Heavy metal exposure in children has been associated with a variety of physiological and neurological problems. The goal of this study was to utilize proteomics to enhance the understanding of biochemical interactions responsible for the health problems related to lead and mercury exposure at concentrations well below CDC guidelines. Blood plasma and serum samples from 34 children were depleted of their most abundant proteins using antibody-based affinity columns and analyzed using two different methods, LC-MS/MS and 2-D electrophoresis coupled with MALDI-TOF/MS and tandem mass spectrometry. Apolipoprotein E demonstrated an inverse significant association with lead concentrations (average being one microgram/deciliter) as deduced from LC-MS/MS and 2-D electrophoresis and confirmed by Western blot analysis. This coincides with prior findings that Apolipoprotein E genotype moderates neurobehavioral effects in individuals exposed to lead. Fifteen other proteins were identified by LC-MS/MS as proteins of interest exhibiting expressional differences in the presence of environmental lead and mercury.