High exposure to statins decrease the risk of new-onset dementia: A nationwide population-based longitudinal cohort study.

Several studies have shown that statin users have a lower risk of new-onset dementia (NOD) compared nonusers. However, other studies have shown opposite results. In this study, we investigated the association between the use of statins and the development of NOD.This was a longitudinal cohort study using data from claim forms submitted to the Taiwanese Bureau of National Health Insurance. The study included patients with NOD and non-NOD subjects from January 2002 to December 2013. We estimated the hazard ratios (HRs) of NOD associated with statin use, whereas nonuser subjects were used as a reference group.A total of 19,522 NOD cases were identified in 100,610 hyperlipidemic patients during the study period. The risk of NOD, after adjusting for sex, age, comorbidities, and concurrent medication, was lower among statin users than nonusers (HR 0.95, 95% CI [confidence interval] 0.94-0.96; P < .001). The adjusted HRs for NOD were 1.53 (95% CI, 1.45-1.62), 0.63 (95% CI, 0.57-0.71), and 0.34 (95% CI, 0.30-0.38) when the cumulative defined daily doses ranged from 28 to 365, 366 to 730, and more than 730 relative to nonusers, respectively.We concluded that statin use is associated with a decreased NOD risk. The protective effect of statins for NOD seemed to be related to high exposure to statins. This study also highlights that high exposure to statins has a dose-response effect on lowering NOD risk.

Meeting report on protein particles and immunogenicity of therapeutic proteins: filling in the gaps in risk evaluation and mitigation.

This meeting was successful in achieving its main goals: (1) summarize currently available information on the origin, detection, quantification and characterization of sub-visible particulates in protein products, available information on their clinical importance, and potential strategies for evaluating and mitigating risk to product quality, and (2) foster communication among academic, industry, and regulatory scientists to define the capabilities of current analytical methods, to promote the development of improved methods, and to stimulate investigations into the impact of large protein aggregates on immunogenicity. There was a general consensus that a considerable amount of interesting scientific information was presented and many stimulating conversations were begun. It is clear that this aspect of protein characterization is in its initial stages. As the development of these new methods progress, it is hoped that they will shed light on the role of protein particulates on product quality, safety, and efficacy. A topic which seemed appropriate for short term follow up was to hold further discussions concerning the development and preparation of one or more standard preparations of protein particulates. This would be generally useful to facilitate comparison of results among different studies, methods, and laboratories, and to foster further development of a common understanding among laboratories and health authorities which is essential to making further progress in this emerging field.

On developing a process for conducting extractable-leachable assessment of components used for storage of biopharmaceuticals.

Extractables and leachables are product-related impurities that result from product contact with components such as gaskets, stoppers, storage bags, cartridges, and prefilled syringes that are used for processing, storage, and/or delivery of biopharmaceuticals. These impurities are a concern for patients due to potential effects on product quality and safety. It is possible that such an impurity could directly impact the patient or indirectly impact the patient by interacting with the protein therapeutics and forming protein adducts. Adducts and leachables may or may not be detected as product-related impurities in routine stability indicating assays depending on the rigor of the analytical program. The need for the development of a thorough and holistic extractable and leachable program based on risk assessment, review of existing literature, and consolidation of industry best practices is discussed. Standardizing component use within an organization enables streamlining of the extractable-leachable program. Our strategy for an extractable-leachable program is divided into different stages, each stage detailing the activities and the department within the organization that is responsible for execution of these activities. The roles and responsibilities of the key stakeholders are identified. The integration of analytical activities with health-based risk-assessment information into the design of an extractable-leachable program is highlighted.

An analytical system for the characterization of highly heterogeneous mixtures of N-linked oligosaccharides.

A novel system for characterizing complex N-linked oligosaccharide mixtures that uses a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), capillary electrophoresis (CE), and high-performance liquid chromatography (HPLC) has been developed. In this study, oligosaccharides released from recombinant TNK-tPA (tissue plasminogen activator) were derivatized with 5-amino-2-naphthalenesulfonic acid (ANSA). The negative charge imparted by the ANSA label facilitated the analysis of the oligosaccharides by MALDI-TOF MS by allowing the observation of both neutral and sialylated oligosaccharides in a single negative ion mode spectrum. Labeling with ANSA was also determined to be advantageous in the characterization of oligosaccharides by both HPLC and CE. The ANSA label was demonstrated to provide superior resolution over the commonly used label 8-aminopyrene-1,3,6-trisulfonic acid (APTS) in both the CE and HPLC analysis of oligosaccharides. To date, no other labels that enable the analysis of complex oligosaccharide mixtures in a single mass spectral mode, while also enabling high-resolution chromatographic and electrophoretic separation of the oligosaccharides, have been reported. By integrating the structural information obtained by MALDI-TOF MS analysis with the ability of CE and HPLC to discriminate between structural isomers, the complete characterization of complex oligosaccharide mixtures is possible.

Characterization of a complex glycoprotein whose variable metabolic clearance in humans is dependent on terminal N-acetylglucosamine content.

Glycoproteins can be cleared from circulation if they carry oligosaccharide structures that are recognized by specific receptors. High-mannose type and asialo complex oligosaccharides are cleared by the mannose and asialoglycoprotein receptors, respectively. This paper presents the protein and terminal saccharide characterization for nine batches of a glycoprotein developed for pharmaceutical use. Each of these batches was evaluated in human pharmacokinetic (PK) studies, and had similar terminal elimination half-lives, but the initial clearance of this glycoprotein varied between batches. The protein is lenercept, an immunoadhesin comprising the Fc domain of human IgG1 and two tumor necrosis factor (TNF) binding domains derived from the extracellular portion of the TNFR1(p55). Lenercept is manufactured in Chinese hamster ovary (CHO) cells and is extensively N-glycosylated but is devoid of high-mannose glycans. The pharmacokinetic variability between these lots only correlated with terminal N-acetylglucosamine and not with terminal galactose, sialic acid or any polypeptide related parameter. The data emphasize the need for appropriate analytical methods for the characterization of glycoproteins, especially those designed for long half-lives, and show that assessment of the content of all three terminal saccharides is sufficient to ensure consistency of their PK performance properties.

A study in glycation of a therapeutic recombinant humanized monoclonal antibody: where it is, how it got there, and how it affects charge-based behavior.

The glycated form of a basic recombinant humanized monoclonal antibody (rhuMAb) was separated and quantitated by boronate affinity chromatography using optimized shielding reagents. Characterization on the isolated glycated material by peptide mapping analysis, using liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) sequencing techniques, identified eight reactive lysine primary amine sites. The glycation reaction extent was similar among the various reactive sites, ranging from approximately 1 to 12%, and a single histidine residue separated the most and least reactive sites. Boronate chromatography run in a linear gradient mode separated monoglycated rhuMAb from higher order glycated species and indicated that the majority ( approximately 90%) of glycated rhuMAb is monoglycated. Low-level glycation on a heavy chain lysine located within a complementarity-determining region (CDR) did not significantly affect binding activity in potency measurements. The glycated forms also behaved as slightly more acidic than the nonglycated antibody in charge-based separation techniques, observable by capillary isoelectric focusing (cIEF) and ion exchange chromatography (IEC). The boronate column has significantly increased retention of aggregated rhuMAb material under separation conditions optimized for the monomer form. Recombinant protein glycation initially occurred during production in mammalian cell culture, where feed sugar and protein concentrations contribute to the total overall glycation on this antibody product.

Optimization and validation of a quantitative capillary electrophoresis sodium dodecyl sulfate method for quality control and stability monitoring of monoclonal antibodies.

In previous work, a capillary electrophoresis sodium dodecyl sulfate (CE-SDS) method using precolumn labeling and laser-induced fluorescence (LIF) detection was developed at Genentech Inc. as part of the control system for the quality control release of a recombinant monoclonal antibody (rMAb) (Hunt, G.; Nashabeh, W. Anal. Chem. 1999, 71, 2390-2397.). In the current work, a generic and quantitative CE-SDS assay with LIF detection of rMAbs with improved accuracy and precision is described. The implementation of an alkylating step with iodoacetamide and optimization of the incubation temperature and time, in the presence of SDS, greatly decrease any thermally induced fragmentation of nonreduced labeled rMAb samples. In addition, a quantitative study of the effects of sample buffer pH on rMAb fragmentation is also presented. Furthermore, the performance of alternative CE-SDS polymer solutions and instrumentation for quantitative analysis of rMAbs is shown in this article. The validation of this method, under the guidelines of the International Committee on Harmonization (ICH), demonstrates that the assay quantitatively determines the consistency of rMAb manufacture as it relates to size heterogeneity and product purity.

Capillary electrophoresis-mass spectrometry as a characterization tool for therapeutic proteins.

With the increasing use of capillary electrophoresis (CE) in the biotechnology industry, there is a demand for analytical tools and methodology that can be used to characterize CE profiles. This article describes the implementation and optimization of a robust online CE-mass spectrometry (CE-MS) system used for the characterization of several CE assays developed at Genentech Inc. These assays include CE as a complement to reverse-phase peptide mapping for the identification of small peptides eluting in the void volume, profiling N-linked glycopeptide heterogeneity, and determining O-linked site occupancy. In addition, CE-MS was used to confirm major 8-aminopyrene-1,3,6-trisulfonate (APTS)-labeled glycans released from recombinant antibodies that are routinely profiled by CE-laser-induced fluorescence (CE-LIF). For each study, CE-MS was able to successfully identify components seen in UV or LIF electropherograms, thereby expanding the capability of CE and CE-MS for profiling biomolecules.