A generic platform to couple affinity chromatography with native mass spectrometry for the analysis of therapeutic monoclonal antibodies.

Affinity chromatography coupled with native mass spectrometry has emerged as a powerful tool for the analysis of therapeutic monoclonal antibodies (mAbs). Exploiting the specific interactions between mAbs and their ligands, these methods not only provide orthogonal means to study the highly complex mAb attributes, but also offer insights on their biological relevance. Despite the great promise, application of affinity chromatography - native mass spectrometry in routine mAb characterization has been limited, largely due to the complicated experimental set up. In this study, we introduced a generic platform to facilitate the online coupling of different affinity separation modes with native mass spectrometry. Built upon a recently introduced native LC-MS platform, this new strategy can accommodate a wide range of chromatographic conditions, and therefore, allow greatly simplified experimental set up and facile swapping of affinity separation modes. The utility of this platform was demonstrated by successful online coupling of three affinity chromatography methods (protein A, FcγRIIIa, and FcRn) with native mass spectrometry. The developed protein A-MS method was tested both in a "bind-and-elute" mode for rapid mAb screening and in a high-resolution resolving mode to study mAb species with altered protein A affinity. The FcγRIIIa-MS method was applied to achieve glycoform-resolved analyses of both IgG1 and IgG4 subclass molecules. The FcRn-MS method was demonstrated in two case studies, where specific post-translational modifications and Fc mutations were known to alter FcRn affinities.

Coupling Anion Exchange Chromatography with Native Mass Spectrometry for Charge Heterogeneity Characterization of Monoclonal Antibodies.

Despite the recent success of coupling anion exchange chromatography with native mass spectrometry (AEX-MS) to study anionic proteins, the utility of AEX-MS methods in therapeutic monoclonal antibody (mAb) characterization has been limited. In this work, we developed and optimized a salt gradient-based AEX-MS method and explored its utility in charge variant analysis of therapeutic mAbs. We demonstrated that, although the developed AEX-MS method is less useful for IgG1 molecules that have higher isoelectric points (pIs), it is an attractive alternative for charge variant analysis of IgG4 molecules. By elevating the column temperature and lowering the mAb pI through PNGase F-mediated deglycosylation, the chromatographical resolution from AEX separation can be significantly improved. We also demonstrated that, after PNGase F and IdeS digestion, the AEX-MS method exhibited excellent resolving power for multiple attributes in the IgG4 Fc region, including unprocessed C-terminal Lys, N-glycosylation occupancy, and several conserved Fc deamidations, making it ideally suited for multiple attribute monitoring (MAM). Through fractionation and peptide mapping analysis, we also demonstrated that the developed AEX-MS method can provide site-specific and isoform-resolved separation of Fc deamidation products, allowing rapid and artifact-free quantitation of these modifications without performing bottom-up analysis.

Post-Column Denaturation-Assisted Native Size-Exclusion Chromatography-Mass Spectrometry for Rapid and In-Depth Characterization of High Molecular Weight Variants in Therapeutic Monoclonal Antibodies.

The high molecular weight (HMW) size variants present in therapeutic monoclonal antibody (mAb) samples need to be closely monitored and characterized due to their impact on product safety and efficacy. Because of the complexity and often low abundances in final drug substance (DS) samples, characterization of such HMW species is challenging and traditionally requires offline enrichment of the HMW species followed by analysis using various analytical tools. Here, we report the development of a postcolumn denaturation-assisted native SEC-MS method that allows rapid and in-depth characterization of mAb HMW species directly from unfractionated DS samples. This method not only provides high-confidence identification of HMW complexes based on accurate mass measurement of both the intact assembly and the constituent subunits but also allows in-depth analysis of the interaction nature and location. In addition, using the extracted ion chromatograms, derived from high-quality, native-like mass spectra, the elution profiles of each noncovalent and/or nondissociable complex can be readily reconstructed, facilitating the comprehension of a complex HMW profile. The utility of this novel method was demonstrated in different applications, ranging from enriched HMW characterization at late stage development, comparability assessment due to process changes, and forced degradation study of coformulated mAbs. As this method does not require prior enrichment, it is thus desirable for providing both rapid and in-depth characterization of HMW species during the development of therapeutic mAbs.

Characterization of Adeno-Associated Virus Capsid Proteins Using Hydrophilic Interaction Chromatography Coupled with Mass Spectrometry.

To support adeno-associated virus (AAV)-based gene therapy development, characterization of the three capsid viral proteins (VP; VP1/VP2/VP3) from recombinant AAV can offer insights on capsid identity, heterogeneity, and product and process consistency. Intact protein mass analysis is a rapid, reliable, and sensitive method to confirm AAV serotypes based on accurate mass measurement of the constituent capsid proteins. Compared to commonly applied reversed-phase liquid chromatography (RPLC) methods, we demonstrated that, using a wide-pore amide-bonded column, hydrophilic interaction chromatography (HILIC) could achieve improved separation of VPs from a variety of AAV serotypes using a generic method prior to MS detection. Moreover, HILIC-based separation was shown to be particularly sensitive in detecting capsid protein variants resulting from different post-translational modifications (PTMs) (e.g. phosphorylation and oxidation) and protein backbone clippings, making it ideally suited for capsid heterogeneity characterization. To overcome the challenges associated with low protein concentrations of AAV samples, as well as the trifluoroacetic acid (TFA)-induced ion suppression during HILIC-MS analysis, different strategies were implemented to improve method sensitivity, including increasing the HILIC column loading and the application of a desolvation gas modification device. Finally, we demonstrated that this integrated HILIC-FLR-MS method can be generically applied to characterize a variety of AAV serotype samples at low concentrations without any sample treatment to achieve unambiguous serotype identification, stoichiometry assessment, and PTM characterization.

An 18O-Labeling Assisted LC-MS Method for Accurate Quantitation of Unprocessed C-Terminal Lysine in Therapeutic Monoclonal Antibodies.

Unprocessed C-terminal lysine (C-term Lys) is one of the most common causes for the formation of basic variants in therapeutic monoclonal antibodies (mAbs). Although the C-term Lys variants are routinely quantified by a LC-MS-based peptide mapping method using the relative MS responses from both C-terminal peptides (with and without Lys), this approach often leads to overestimation of Lys-containing peptide due to the intrinsic difference in ionization efficiency. Herein, we report an 18O-labeling assisted LC-MS method, which takes advantage of the carboxypeptidase B-catalyzed Lys removal and 18O-labeling to achieve improved accuracy of C-term Lys quantitation. The fidelity of this method was first demonstrated using synthetic peptide mixture standards that mimic a wide range of C-term Lys levels. Finally, the newly developed method was applied in a case study where C-term Lys variants in mAb samples manufactured from different processes were accurately quantified and compared. This new method provides a valuable solution for studies where accurate C-term Lys levels are needed to assist decision-making and root-cause investigation.

Simple Approach for Improved LC-MS Analysis of Protein Biopharmaceuticals via Modification of Desolvation Gas.

LC-MS based analysis of protein biopharmaceuticals could benefit from improved data quality, which can subsequently lead to improved drug characterization with higher confidence and less ambiguity. In this study, we created a simple device to modify the desolvation gas on a Q-Exactive mass spectrometer and to demonstrate the utility in improving both peptide mapping analysis and intact mass analysis, the two most routinely and widely applied LC-MS techniques in protein biopharmaceutical characterization. By modifying the desolvation gas with acid vapor from propionic acid (PA) and isopropanol (IPA), the ion suppression effects from trifluoroacetic acid (TFA) in a typical peptide mapping method can be effectively mitigated, thus leading to improved MS sensitivity. By modifying the desolvation gas with base vapor from triethylamine (TEA), the charge reduction effect can be achieved and utilized to improve the spectral quality from intact mass analysis of protein biopharmaceuticals. The approach and device described in this work suggests a low-cost and practical solution to improve the LC-MS characterization of protein biopharmaceuticals, which has the potential to be widely implemented in biopharmaceutical analytical laboratories.

Ultrasensitive Characterization of Charge Heterogeneity of Therapeutic Monoclonal Antibodies Using Strong Cation Exchange Chromatography Coupled to Native Mass Spectrometry.

In therapeutic monoclonal antibody (mAb) development, charge heterogeneity of a mAb molecule is often associated with critical quality attributes and is therefore monitored throughout development and during QC release to ensure product and process consistency. Elucidating the cause of each charge variant species is an involved process that often requires offline fractionation by ion exchange chromatography (IEX) followed by mass spectrometry (MS) analysis, largely due to the incompatibility of conventional IEX buffers for direct MS detection. In this study, we have developed a method that combines a generic strong cation exchange (SCX) chromatography step with ultrasensitive online native MS analysis (SCX-MS) optimized for mAb separation and detection. As demonstrated by analyzing mAb molecules with a wide range of pI (isoelectric point) values, the developed method can consistently achieve both high-resolution IEX separation and ultrasensitive MS detection of low-abundance charge variant species. Using this method, we analyzed the charge heterogeneity of NISTmAb reference material 8671 (NISTmAb) at both whole antibody and subdomain levels. In particular, due to the high sensitivity, a nonconsensus Fab glycosylation site, present at a very low level (<0.1%), was directly detected in the NISTmAb sample without any enrichment. The structure and location of this Fab glycosylation was further characterized by peptide mapping analysis. Despite the extensive characterization of NISTmAb material in previous studies, this is the first time that this Fab-glycosylated variant has been identified in the NISTmAb, demonstrating the value of this new method in achieving a more comprehensive characterization of charge heterogeneity for therapeutic mAbs.

Characterization of product-related low molecular weight impurities in therapeutic monoclonal antibodies using hydrophilic interaction chromatography coupled with mass spectrometry.

Traditional SDS-PAGE method and its modern equivalent CE-SDS method are both widely applied to assess the purity of therapeutic monoclonal antibody (mAb) drug products. However, structural identification of low molecular weight (LMW) impurities using those methods has been challenging and largely based on empirical knowledges. In this paper, we present that hydrophilic interaction chromatography (HILIC) coupled with mass spectrometry analysis is a novel and orthogonal method to characterize such LMW impurities present within a purified mAb drug product sample. We show here that after removal of N-linked glycans, the HILIC method separates mAb-related LMW impurities with a size-based elution order. The subsequent mass measurement from a high-resolution accurate mass spectrometer provides direct and unambiguous identification of a variety of low-abundance LMW impurities within a single LC-MS analysis. Free light chain, half antibody, H2L species (antibody possessing a single light chain) and protein backbone-truncated species can all be confidently identified and elucidated in great detail, including the truncation sites and associated post-translational modifications. It is worth noting that this study provides the first example where the H2L species can be directly detected in a mAb drug product sample by intact mass analysis without prior enrichment.

Effects of neighborhood building density, height, greenspace, and cleanliness on indoor environment and health of building occupants.

The influences of indoor environment quality on occupant health have long been one of the main focuses in built environment and public health research. However, evidence to this effect has been inconsistent. Furthermore, previous urban studies have indicated the interaction between urban morphology and indoor environment. This study thus goes beyond indoor environment to investigate: i) the effects of neighborhood environment on occupant health; and ii) the mediating roles of indoor environment on the neighborhood environment and occupant health relationships. To achieve this aim, buildings located in different neighborhood environment in Hong Kong are selected. Data are collected by post-occupancy evaluation (occupant health), indoor environment assessment (thermal comfort, indoor air quality, ventilation, visual comfort, and acoustic comfort) and neighborhood environment assessment (neighborhood building density, building height, cleanliness and greenspace) through questionnaire survey. Through correlation analysis, regression modelling and Sobel test, it is found that: i) occupant health is significantly affected by neighborhood building height, building density and cleanliness; ii) the relationships between neighborhood environment and occupant health are significantly mediated by indoor environment, in terms of visual and acoustic comfort; and iii) neighborhood greenspace affects occupant health indirectly through influencing indoor air quality. To cross validate the results of the survey study, which is conducted using subjective data, objective measurements and analyses are further conducted. The objective study, echoing the survey study results, indicates that buildings with lower neighborhood building density and height, and cleaner neighborhood environment have better visual (higher illuminance level) and acoustic (lower noise level) performances.

The Pex1-G844D mouse: a model for mild human Zellweger spectrum disorder.

Zellweger spectrum disorder (ZSD) is a disease continuum that results from inherited defects in PEX genes essential for normal peroxisome assembly. These autosomal recessive disorders impact brain development and also cause postnatal liver, adrenal, and kidney dysfunction, as well as loss of vision and hearing. The hypomorphic PEX1-G843D missense allele, observed in approximately 30% of ZSD patients, is associated with milder clinical and biochemical phenotypes, with some homozygous individuals surviving into early adulthood. Nonetheless, affected children with the PEX1-G843D allele have intellectual disability, failure to thrive, and significant sensory deficits. To enhance our ability to test candidate therapies that improve human PEX1-G843D function, we created the novel Pex1-G844D knock-in mouse model that represents the murine equivalent of the common human mutation. We show that Pex1-G844D homozygous mice recapitulate many classic features of mild ZSD cases, including growth retardation and fatty livers with cholestasis. In addition, electrophysiology, histology, and gene expression studies provide evidence that these animals develop a retinopathy similar to that observed in human patients, with evidence of cone photoreceptor cell death. Similar to skin fibroblasts obtained from ZSD patients with a PEX1-G843D allele, we demonstrate that murine cells homozygous for the Pex1-G844D allele respond to chaperone-like compounds, which normalizes peroxisomal ß-oxidation. Thus, the Pex1-G844D mouse provides a powerful model system for testing candidate therapies that address the most common genetic cause of ZSD. In addition, this murine model will enhance studies focused on mechanisms of pathogenesis.

Newborn screening for X-linked adrenoleukodystrophy: further evidence high throughput screening is feasible.

X-linked adrenoleukodystrophy (ALD) is characterized by adrenal insufficiency and neurologic involvement with onset at variable ages. Plasma very long chain fatty acids are elevated in ALD; even in asymptomatic patients. We demonstrated previously that liquid chromatography tandem mass spectrometry measuring C26:0 lysophosphatidylcholine reliably identifies affected males. We prospectively applied this method to 4689 newborn blood spot samples; no false positives were observed. We show that high throughput neonatal screening for ALD is methodologically feasible.

Mapping of discontinuous conformational epitopes by amide hydrogen/deuterium exchange mass spectrometry and computational docking.

Understanding antigen-antibody interactions at the sub-molecular level is of particular interest for scientific, regulatory, and intellectual property reasons, especially with increasing demand for monoclonal antibody therapeutic agents. Although various techniques are available for the determination of an epitope, there is no widely applicable, high-resolution, and reliable method available. Here, a combination approach using amide hydrogen/deuterium exchange coupled with proteolysis and mass spectrometry (HDX-MS) and computational docking was applied to investigate antigen-antibody interactions. HDX-MS is a widely applicable, medium-resolution, medium-throughput technology that can be applied to epitope identification. First, the epitopes of cytochrome c-E8, IL-13-CNTO607, and IL-17A-CAT-2200 interactions identified using the HDX-MS method were compared with those identified by X-ray co-crystal structures. The identified epitopes are in good agreement with those identified using high-resolution X-ray crystallography. Second, the HDX-MS data were used as constraints for computational docking. More specifically, the non-epitope residues of an antigen identified using HDX-MS were designated as binding ineligible during computational docking. This approach, termed HDX-DOCK, gave more tightly clustered docking poses than stand-alone docking for all antigen-antibody interactions examined and improved docking results significantly for the cytochrome c-E8 interaction.

Newborn screening for X-linked adrenoleukodystrophy (X-ALD): validation of a combined liquid chromatography-tandem mass spectrometric (LC-MS/MS) method.

Newborn screening for X-linked adrenoleukodystrophy (X-ALD) has until now been limited in implementation because of the lack of an accepted standard methodology. We have previously reported a technique using LC-MS/MS analysis that could provide the basis for screening of newborns for X-ALD. The target analyte diagnostic for X-ALD and other peroxisomal disorders of peroxisomal beta-oxidation is 1-hexacosanoyl-2-lyso-sn-3-glycero-phosphorylcholine (26:0-lyso-PC). We report here the validation of the analytical method using an authentic standard of the target compound. The method possesses sensitivity of <1.0fmole injected on column with a correlation coefficient (R(2)) of 0.9987. A tetradeuterated analog of 26:0-lyso-PC served as the internal standard. The sensitivity of this clinical method was confirmed using 17 newborn samples of individuals with peroxisomal disorders retrieved from state newborn screening programs. These samples were run masked with over 1000 newborn samples. All affected individuals were identified with one exception. One sample which was retrieved as an affected did not have the biochemical or genetic abnormality of X-ALD and thus is considered an error in sample identity. These studies clearly show that the method is highly sensitive and accurate in identifying individuals with a defect in peroxisomal beta-oxidation such as X-ALD.

Long-term cognitive outcome in moderate to severe traumatic brain injury: a meta-analysis examining timed and untimed tests at 1 and 4.5 or more years after injury.

OBJECTIVES: To examine long-term outcome of moderate to severe traumatic brain injury (TBI) on timed and untimed cognitive tests using meta-analysis. DESIGN: Meta-analysis examining outcome at 2 epochs, 6 to 18 months postinjury (epoch 1) and 4.5 to 11 years postinjury (epoch 2). SETTING: Data source was published articles (1966-2007) identified through electronic and manual search. PARTICIPANTS: A total of 1380 subjects with moderate to severe TBI participated in the 16 studies meeting inclusion criteria. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Timed and untimed neuropsychologic tests with quantitative results (means, SDs, t, and df tests) from studies containing a healthy comparison group and a mean time since injury falling within 1 of the 2 epochs. RESULTS: Patient versus control weighted effect sizes were medium to large at epoch 1 for both untimed tasks (r=-.46; confidence interval [CI], -.32 to -.65) and timed tasks (r=-.46; CI, -.35 to -.59). At epoch 2, effect sizes were slightly smaller for untimed tasks (r=-.38; CI, -.25 to -.60) and timed tasks (r=-.40; CI, -.32 to -.62). CONCLUSIONS: Patients showed robust, persisting impairments on both timed and untimed tests at recovery plateau (ie, 6-18mo postinjury) and many years later. These findings converge with previous studies, though using an alternative approach that obviates some of the methodologic problems of longitudinal studies, such as selective attrition.

Combined liquid chromatography-tandem mass spectrometry as an analytical method for high throughput screening for X-linked adrenoleukodystrophy and other peroxisomal disorders: preliminary findings.

Utilizing combined liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the analytical method, we have demonstrated a ten to sixtyfold excess of lysophosphatidyl choline containing hexacosanoic acid (26:0) in dried blood spots on a filter paper matrix from 25 male patients with X-linked adrenoleukodystrophy and nine patients with peroxisome biogenesis disorders compared to 19 controls. There was no overlap between normal subjects versus affected subjects.

Ethics in construction project briefing.

The research reported in this paper set out to investigate ethics in the initial stages of construction projects. Briefing is the first real contact stage between the commissioner (client/employer) of a project--at this stage a potential project--and those involved in project realization--the designers and, subsequently, the constructors. It is well known that early decisions are of greatest impact and so, the importance of the initial contacts, communications and consequent decisions are paramount. Different project participants are known to pursue individual objectives to varying degrees as well as possessing different perspectives and perceptions and operating/behaving in different ways. Hence, determination of the appropriate form, content etc. of a project is, inevitably, a matter of exercising value judgements and compromises and so, involves ethical considerations. A case study of a project through the briefing stage is reported and analysed, from initial contacts to scheme approval. It is apparent that a number of ethical concerns are manifest through the various actions of the major participants.

Adverse events and complications among patients admitted to hospital in the home directly from the emergency department.

INTRODUCTION: The nature and incidence of adverse events and complications among patients admitted from the emergency department to hospital in the home has not been investigated. This study aimed to investigate this problem and make recommendations for prevention strategies. METHODS: This was an explicit retrospective chart review of patients admitted from the emergency department directly to hospital in the home between 22 February 1995 and 1 September 2000. A data extraction document was designed specifically for the study and used to extract data relating to patient demographics, diagnosis, past medical history and outcome. The outcomes of interest include adverse events, complications and death. An adverse effect is defined as an unintended injury or complication that results in disability, death or prolonged hospital stay and is caused by health care management. These adverse events may occur prior to or during the index admission and may be noted during or after the index admission. A complication is defined as an undesirable outcome that occurs during the management but not causing disability, death or prolonged hospital stay. RESULTS: Three hundred and fifty-seven patients were enrolled (51.3% male; median age 52 years, range 16-96 years). Fifty-five adverse events were identified: 49 adverse events (89%) were due to management prior to hospital in the home admission and six adverse events (10.9%) were directly attributable to hospital in the home management. This represents a rate of 1.7 adverse events per 100 hospital in the home admissions directly attributable to hospital in the home management. One hundred and eighteen complications were identified. Most complications were easily managed. Thirty-one patients had unplanned re-admissions and two patients died within 28 days of hospital in the home admission. CONCLUSION: Most patients admitted to hospital in the home from the emergency department were managed successfully. Few adverse events arose from hospital in the home treatment. Complications were common but minor in nature. Strategies for the prevention of phlebitis and constipation are recommended.