Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

Immunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fc-glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans.     

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 2: Mass spectrometric methods

To monitor the Fc glycosylation of therapeutic immunoglobulin G in bioprocess development, product characterization and release analytics, reliable techniques for glycosylation analysis are needed. Several analytical methods are suitable for this application. We recently presented results comparing detection methods for glycan analysis that are separation-based, but did not include mass spectrometry (MS). In the study reported here, we comprehensively compared MS-based methods for Fc glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods investigated were compared with respect to precision, accuracy, throughput and analysis time. Emphasis was put on the detection and quantitation of sialic acid-containing glycans. Eleven MS methods were compared to hydrophilic interaction liquid chromatography of 2-aminobenzamide labeled glycans with fluorescence detection, which served as a reference method and was also used in the first part of the study. The methods compared include electrospray MS of the heavy chain and Fc part after limited digestion, liquid chromatography MS of a tryptic digest, porous graphitized carbon chromatography MS of released glycans, electrospray MS of glycopeptides, as well as matrix assisted laser desorption ionization MS of glycans and glycopeptides. Most methods showed excellent precision and accuracy. Some differences were observed with regard to the detection and quantitation of low abundant glycan species like the sialylated glycans and the amount of artefacts due to in-source decay.     

Comparison of the volatile emission profiles of ground almond and pistachio mummies: Part 1 – Addressing a gap in knowledge of current attractants for navel orangeworm

Over the years various tissues of almond and pistachio have been evaluated for their ability to attract the navel orangeworm moth, a major insect pest in California tree nut orchards. Almond meal, which typically consists of ground almond kernels, is a monitoring tool for navel orangeworm populations in almond and pistachio orchards. Recently, ground pistachio and almond mummies have been re-visited in field trapping studies for their potential to attract navel orangeworm moths. Surprisingly, the volatile profiles of these two systems have not been reported. The objective of this study was to survey and then compare and contrast the volatile profiles of both ground almond and pistachio mummies over the course of a week. Principal component analysis (PCA) of the headspace volatiles showed a distinct difference between ground almond and pistachio mummies. The volatile emission profile of the almond mummies remained consistent over the one-week period, albeit in low content and composition; whereas the profile of the pistachio mummies changed over time as shown by PCA. Seven compounds were identified as being common volatiles to both matrices. These data may help explain results from recent navel orangeworm field trapping studies using almond and pistachio mummies, as well as with the formulation of future synthetic blends.     

Exhaled breath analysis: a review of ‘breath-taking’ methods for off-line analysis

Background

The potential of exhaled breath sampling and analysis has long attracted interest in the areas of medical diagnosis and disease monitoring. This interest is attributed to its non-invasive nature, access to an unlimited sample supply (i.e., breath), and the potential to facilitate a rapid at patient diagnosis. However, progress from laboratory setting to routine clinical practice has been slow. Different methodologies of breath sampling, and the consequent difficulty in comparing and combining data, are considered to be a major contributor to this. To fulfil the potential of breath analysis within clinical and pre-clinical medicine, standardisation of some approaches to breath sampling and analysis will be beneficial.

Objectives

The aim of this review is to investigate the heterogeneity of breath sampling methods by performing an in depth bibliometric search to identify the current state of art in the area. In addition, the review will discuss and critique various breath sampling methods for off-line breath analysis.

Methods

Literature search was carried out in databases MEDLINE, BIOSIS, EMBASE, INSPEC, COMPENDEX, PQSCITECH, and SCISEARCH using the STN platform which delivers peer-reviewed articles. Keywords searched for include breath, sampling, collection, pre-concentration, volatile. Forward and reverse search was then performed on initially included articles. The breath collection methodologies of all included articles was subsequently reviewed.

Results

Sampling methods differs between research groups, for example regarding the portion of breath being targeted. Definition of late expiratory breath varies between studies.

Conclusions

Breath analysis is an interdisciplinary field of study using clinical, analytical chemistry, data processing, and metabolomics expertise. A move towards standardisation in breath sampling is currently being promoted within the breath research community with a view to harmonising analysis and thereby increasing robustness and inter-laboratory comparisons.

     

Comparison of derivatization and chromatographic methods for GC–MS analysis of amino acid enantiomers in physiological samples

GC–MS analysis of fluorinated and non-fluorinated chloroformate and anhydride derivatives of amino acid (AA) enantiomers on two different chiral columns was compared for the direct quantification of free l- and d-AAs in human serum and urine in a single analytical run. Best sensitivity was achieved with pentafluoropropionic anhydride/heptafluorobutanol derivatives separated on a Chirasil-l-Val column. However, the occurrence of racemization during derivatization precluded accurate quantification of AA enantiomers. Derivatization with methyl chloroformate/methanol and separation on an Rt-γDEXsa column did not exhibit racemization and yielded ten baseline separated racemates of proteinogenic AAs with resolution values greater than 2.4. However, protein and peptide hydrolysis occurred in serum and urine during the highly exothermal derivatization reaction under alkaline conditions. Removing serum proteins by precipitation before derivatization and performing the reaction at neutral pH enabled the determination of accurate free AA enantiomer concentrations. Accuracy of quantification was validated by an established nonchiral GC–MS method for AA analysis. Reliable quantification was achieved using stable-isotope labeled l-AAs as internal standards. Limits of detection (LOD) and lower limits of quantification (LLOQ) for the d-AAs were in the range of 3.2–446 nM and 0.031–1.95 μM, respectively. Relative standard deviations (N = 6) for the measurement of AAs in urine and serum ranged from 0.49–11.10% to 0.70–3.87%, respectively. The method was applied to the analysis of urine from 19 patients with renal insufficiency. In comparison to healthy probands, D-ratios of Ala, Val, Pro, Thr, Asp, and Asn were significantly increased.     

Guardians at the gate: Patent protection for therapeutic monoclonal antibodies—part 1

Patents provide one of the few protections companies can avail themselves of to help protect their therapeutic monoclonal antibody products. Just as the therapeutic monoclonal antibody field is constantly evolving, so too is the legal environment surrounding these inventions. In a series of articles, the general state of the law surrounding therapeutic antibodies will be explained, and important challenges to this technology area will be discussed. Much is at stake when companies market therapeutic monoclonal antibodies; therefore, a firm understanding of this important form of protection is critically important for anyone developing such products.Key words: patent, therapeutic, monoclonal antibodies, KSR, life cycle management     

Product Quality Research Institute evaluation of cascade impactor profiles of pharmaceutical aerosols: Part 2—Evaluation of a method for determining equivalence

The purpose of this article is to present the thought process, methods, and interim results of a PQRI Working Group, which was charged with evaluating the chi-square ratio test as a potential method for determining in vitro equivalence of aerodynamic particle size distribution (APSD) profiles obtained from cascade impactor measurements. Because this test was designed with the intention of being used as a tool in regulatory review of drug applications, the capability of the test to detect differences in APSD profiles correctly and consistently was evaluated in a systematic way across a designed space of possible profiles. To establish a “base line,” properties of the test in the simplest case of pairs of identical profiles were studied. Next, the test's performance was studied with pairs of profiles, where some difference was simulated in a systematic way on a single deposition site using realistic product profiles. The results obtained in these studies, which are presented in detail here, suggest that the chi-square ratio test in itself is not sufficient to determine equivalence of particle size distributions. This article, therefore, introduces the proposal to combine the chi-square ratio test with a test for impactor-sized mass based on Population Bioequivalence and describes methods for evaluating discrimination capabilities of the combined test. The approaches and results described in this article elucidate some of the capabilities and limitations of the original chi-square ratio test and provide rationale for development of additional tests capable of comparing APSD profiles of pharmaceutical aerosols. Published: January 19, 2007     

Nrf2—a therapeutic target for the treatment of neurodegenerative diseases

The brain is very sensitive to changes in redox status; thus maintaining redox homeostasis in the brain is critical for the prevention of accumulating oxidative damage. Aging is the primary risk factor for developing neurodegenerative diseases. In addition to age, genetic and environmental risk factors have also been associated with disease development. The primary reactive insults associated with the aging process are a result of oxidative stress (OS) and nitrosative stress (NS). Markers of increased oxidative stress, protein and DNA modification, inflammation, and dysfunctional proteostasis have all been implicated in contributing to the progression of neurodegeneration. The ability of the cell to combat OS/NS and maintain a clearance mechanism for misfolded aggregating proteins determines whether or not it will survive. A critical pathway in this regard is the Nrf2 (nuclear factor erythroid 2-related factor 2)- antioxidant response element (ARE) pathway. Nrf2 activation has been shown to mitigate a number of pathologic mechanisms associated with Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, Huntington’s disease, and multiple sclerosis. This review will focus on the role of Nrf2 in these diseases and the potential for Nrf2 activation to attenuate disease progression.     

Guardians at the gate: Biosimilar and patent reform legislation could fundamentally change the guards for therapeutic monoclonal antibodies—Part 2

Patent protection and FDA exclusivities are the two principal forms of protection available to companies that develop therapeutic monoclonal antibodies. Propo-sed changes to both forms of protection are currently being debated in the United States Congress. Specifically, Congress is presently debating both biosimilar and patent reform legislations. Although no bill has yet passed, it is expected that patent reform legislation should pass this year. It is less likely that a biosimilar bill will pass this year. However, when legislations are enacted, the changes will significantly impact the business of therapeutic monoclonal antibodies.Key words: patent reform, biosimilar legislation, therapeutic antibody, Eshoo, Waxman, S 615, HR 1260     

Linear response theory: An alternative to PB and GB methods for the analysis of molecular dynamics trajectories?

We explore the use of classical Linear Response Theory (LRT) as an alternative strategy to the use of Molecular Mechanics/Poisson-Boltzmann strategies to compute the solvation free energy of macromolecules from molecular dynamics simulations using an explicit representation of solvent. The method reproduces well the free energy of solvation of standard amino acid side chains, small peptides, and proteins. The use of a fully discrete representation of solvent avoids the possible problems of continuum models to represent the solvation of systems containing tightly bound water molecules.     

Human Vκ immunoglobulin gene number: Implications for the origin of antibody diversity

To assess the relative contributions of germline versus somatically mutated genes in the human immune system, we have examined the size of the kappa light-chain variable region (V_κ) gene pool. Two cloned kappa subgroup 1 (V_κ1) gene probes detected the same family of 15 to 20 crosshybridizing restriction fragments in human DNA, whereas flanking region probes detected fewer hybridizing fragments. Most of the hybridizing bands represent single-copy genes, as judged by a “gene titration” experiment. Furthermore, the number of hybridization bands is a good estimate of the haploid gene number, since we observed little polymorphism of restriction sites in the V_κ locus of eight unrelated people. A cloned V_κ3 probe hybridized to essentially the same 15–20 genes in human DNA as the V_κ1 probes. These results strongly suggest that a discrete family of 15–20 genes constitutes a large proportion of the V genes from three of the four V_κ subgroups. The small number of V_κ genes in the human genome supports the idea that somatic mutation plays a major role in the origin of antibody diversity in man.     

Comparison of different methods for the isolation of mesenchymal stem cells from human umbilical cord Wharton’s jelly

Several techniques have been devised for the dissociation of tissues for primary culture. These techniques can affect the quantity and quality of the isolated cells. The aim of our study was to develop the most appropriate method for the isolation of human umbilical cord-derived mesenchymal (hUCM) cells. In the present study, we compared four methods for the isolation of hUCM cells: three enzymatic methods; collagenase/hyaluronidase/trypsin (CHT), collagenase/trypsin (CT) and trypsin (Trp), and an explant culture (Exp) method. The trypan blue dye exclusion test, the water-soluble tetrazolium salt-1 (WST-1) assay, flow cytometry, alkaline phosphatase activity and histochemical staining were used to evaluate the results of the different methods. The hUCM cells were successfully isolated by all methods but the isolation method used profoundly altered the cell number and proliferation capacity of the isolated cells. The cells were successfully differentiated into adipogenic and osteogenic lineages and alkaline phosphatase activity was detected in the hUCM cell colonies of all groups. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed in all groups, while CD34 and CD45 were not expressed. The expression of C-kit in the enzymatic groups was higher than in the explant group, while the expression of Oct-4 was higher in the CT group compared to the other groups. We concluded that the collagenase/trypsin method of cell isolation yields a higher cell density than the others. These cells expressed a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method of cell isolation resulted in a higher cell proliferation rate and activity compared to the other methods.     

Comparison of two kinds of methods to determine the titer of recombinant retrovirus containing β-globin gene based on G418 selection

Four recombinant retrovirus (RV) vectors containing human β-globin gene and regulatory elements were constructed. To determine the titers of recombinant RV from corresponding producer cell lines, we compared two kinds of method (the simple and the conventional) based on G418 resistance, which is conferred by neo gene of RV vector. The results demonstrated that the simple method shortened the selection period to 3 d instead of 10–12 d with the conventional method and reduced the amount of work; importantly, the titers determined by the simple method were not different significantly from those measured by the conventional method. It can be concluded that the simple method can be used to determine the titers of recombinant RV containing not only cDNA but also genomic DNA with introns and complex regulatory elements instead of the conventional method.     

Simple methods for the 3′ biotinylation of RNA

Biotinylation of RNA allows its tight coupling to streptavidin and is thus useful for many types of experiments, e.g., pull-downs. Here we describe three simple techniques for biotinylating the 3′ ends of RNA molecules generated by chemical or enzymatic synthesis. First, extension with either the Schizosaccharomyces pombe noncanonical poly(A) polymerase Cid1 or Escherichia coli poly(A) polymerase and N6-biotin-ATP is simple, efficient, and generally applicable independently of the 3′-end sequences of the RNA molecule to be labeled. However, depending on the enzyme and the reaction conditions, several or many biotinylated nucleotides are incorporated. Second, conditions are reported under which splint-dependent ligation by T4 DNA ligase can be used to join biotinylated and, presumably, other chemically modified DNA oligonucleotides to RNA 3′ ends even if these are heterogeneous as is typical for products of enzymatic synthesis. Third, we describe the use of ϕ29 DNA polymerase for a template-directed fill-in reaction that uses biotin-dUTP and, thanks to the enzyme''s proofreading activity, can cope with more extended 3′ heterogeneities.     

Effect of the light chain C-terminal serine residue on disulfide bond susceptibility of human immunoglobulin G1λ

The light chain cysteine residue that forms an interchain disulfide bond with the cysteine residue in the heavy chain in IgG1κ is the last amino acid. The cysteine residue is followed by a serine residue in IgG1λ. Effect of the serine residue on the susceptibility of disulfide bonds to reduction was investigated in the current study using a method including reduction, differential alkylation using iodoacetic acid with either natural isotopes or enriched with carbon-13, and mass spectrometry analysis. This newly developed method allowed an accurate determination of the susceptibility of disulfide bonds in IgG antibodies. The effect of the serine residue on disulfide bond susceptibility was compared using three antibodies with differences only in the light chain last amino acid, which was either a serine residue, an alanine residue or deleted. The results demonstrated that the presence of the amino acid (serine or alanine) increased the susceptibility of the inter light and heavy chain disulfide bonds to reduction. On the other hand, susceptibility of the two inter heavy chain disulfide bonds and intrachain disulfide bonds was not changed significantly.     

Comparison of methods for detecting vulnerability of xylem embolism in Robinia pseudoacacia北大核心CSCD

Aims The vulnerability of xylem embolism is one of the key physiological factors that are related to plant mortality. Vulnerability curves are typically used for determining the vulnerability of xylem embolism. However, the shapes of vulnerability curves vary with the methods of assessment, especially in plant species with long xylem vessels. This study aims to investigate the feasibility of using different methods for establishment of vulnerability curves. Methods Robinia pseudoacacia branches, with long xylem vessels, were used as plant materials for comparison of three different methods in establishing vulnerability curves, including bench top dehydration, Cochard Cavitron centrifugation and Sperry centrifugation. In the Sperry centrifugation method, rotors of two different sizes were used to test the ‘open vessel artifact’ hypothesis. Important findings The vulnerability curve established by the bench top dehydration method displayed an “s” shape, while both the Cochard Cavitron centrifugation and Sperry centrifugation methods produced “r” shape curves. Vulnerability curves derived from the bench top dehydration method and the centrifugation methods were significantly different. Using the Sperry centrifugation method, the R. pseudoacacia branch samples in the 14.4 cm rotor had a higher proportion of open vessels, while the embolic vulnerability curves established on the 27.4 cm and 14.4 cm long stem segments were similar, indicating that the Sperry centrifugation method does not produce “open vessel artifact”. © Chinese Journal of Plant Ecology.     

Comparison of ‘ignition’ and ‘extraction’ methods for the determination of organic phosphate in rocks and soils

Summary Ignition markedly increased the solubility in dilute acid of iron- and aluminium-bound inorganic phosphate in samples of weathered New Zealand greywacke rock. This observation supports the view that ignition methods may sometimes overestimate the total organic phosphate of soils.