The size, heterogeneity, and biological production process of protein therapeutics like monoclonal antibodies create unique challenges for their analysis and regulation compared with small molecules. ...Complete structural characterization of a molecule 1000-fold heavier than aspirin is no small feat. Biological post-translational modifications such as glycosylation further complicate their characterization and regulation. Even approved protein therapeutics are known to contain multiple structural variants in differing amounts. Structural modification occurs during production and storage as well as within patients after administration. Thus, the goals of manufacturers and regulators are to control and characterize this heterogeneity, not take on the impossible task of eliminating it. The aim of this review is to describe the structural heterogeneities known to occur with immunoglobulin G (IgG), note current detection and analytical strategies, establish their causes, and define their potential effects on the ultimate safety, purity, and potency of antibody therapeutics when known.
We hypothesized that there is decreased synthesis of glutathione (GSH) in type 2 diabetes (T2DM) especially in the presence of microvascular complications, and this is dependent on the degree of ...hyperglycemia.
In this case-control study, we recruited 16 patients with T2DM (7 without and 9 with microvascular complications), and 8 age- and sex-matched non-diabetic controls. We measured GSH synthesis rate using an infusion of 2H2-glycine as isotopic tracer and collection of blood samples for liquid chromatography mass spectrometric analysis.
Compared to the controls, T2DM patients had lower erythrocyte GSH concentrations (0.90 ± 0.42 vs. 0.35 ± 0.30 mmol/L; P = 0.001) and absolute synthesis rates (1.03 ± 0.55 vs. 0.50 ± 0.69 mmol/L/day; P = 0.01), but not fractional synthesis rates (114 ± 45 vs. 143 ± 82%/day; P = 0.07). The magnitudes of changes in patients with complications were greater for both GSH concentrations and absolute synthesis rates (P-values ≤ 0.01) compared to controls. There were no differences in GSH concentrations and synthesis rates between T2DM patients with and without complications (P-values > 0.1). Fasting glucose and HbA1c did not correlate with GSH concentration or synthesis rates (P-values > 0.17).
Compared to non-diabetic controls, patients with T2DM have glutathione deficiency, especially if they have microvascular complications. This is probably due to reduced synthesis and increased irreversible utilization by non-glycemic mechanisms.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Type 1 diabetes (T1D) is an autoimmune disease characterized by T and B cell responses to proteins expressed by insulin-producing pancreatic β cells, inflammatory lesions within islets (insulitis), ...and β cell loss. We previously showed that Ag-specific tolerance targeting single β cell protein epitopes is effective in preventing T1D induced by transfer of monospecific diabetogenic CD4 and CD8 transgenic T cells to NOD.
mice. However, tolerance induction to individual diabetogenic proteins, for example, GAD65 (glutamic acid decarboxylase 65) or insulin, has failed to ameliorate T1D both in wild-type NOD mice and in the clinic. Initiation and progression of T1D is likely due to activation of T cells specific for multiple diabetogenic epitopes. To test this hypothesis, recombinant insulin, GAD65, and chromogranin A proteins were encapsulated within poly(d,l-lactic-
-glycolic acid) (PLGA) nanoparticles (COUR CNPs) to assess regulatory T cell induction, inhibition of Ag-specific T cell responses, and blockade of T1D induction/progression in NOD mice. Whereas treatment of NOD mice with CNPs containing a single protein inhibited the corresponding Ag-specific T cell response, inhibition of overt T1D development only occurred when all three diabetogenic proteins were included within the CNPs (CNP-T1D). Blockade of T1D following CNP-T1D tolerization was characterized by regulatory T cell induction and a significant decrease in both peri-insulitis and immune cell infiltration into pancreatic islets. As we have recently published that CNP treatment is both safe and induced Ag-specific tolerance in a phase 1/2a celiac disease clinical trial, Ag-specific tolerance induced by nanoparticles encapsulating multiple diabetogenic proteins is a promising approach to T1D treatment.
Liquid chromatography-mass spectrometry (LC-MS) is an information rich analytical tool that can provide fast, robust and sensitive characterization of protein therapeutics for quality assurance and ...structural comparison. Herein, structural characterization of two anti-CD20 monoclonal antibodies obtained from two different sources was performed using a middle-down LC-MS strategy to determine if they can be analytically differentiated. Through the use of a specific enzymatic digestion method using IdeS with subsequent LC-MS analysis, we show that the anti-CD20 monoclonal antibody that has been approved by the FDA can be partially characterized and differentiated analytically from an Indian sourced product that lacks FDA approval. In comparison to the FDA-approved product, differential modifications to both the N- and C-termini result in increased charge heterogeneity for the Indian product. In addition, significant differences in the intensities of the observed glycoforms between the two antibodies were detected. While this study assesses only one lot of each of a FDA approved drug product and the Indian sourced drug product, the observed differences may represent process specific fingerprints that could be useful for surveillance purposes.
Middle-down LC-MS analysis can be used to identify structural heterogeneities between a monoclonal antibody from different sources.
This work describes orthogonal NMR and MS tests for the structure and composition of the drug protamine sulfate derived from chum salmon. The spectral response pattern obtained by 1D-
1
H-NMR and MS ...methods from salmon protamine, a mixture of four predominant peptide chains, is dependent on the amino acid sequence and abundance of each peptide. Thus, an assay was developed based on the ratios of alanine, glycine and arginine amino acid residue NMR peaks (relative to the arginine CδH proton signal) in this mixture that are unique to the salmon source. In addition, MS analysis provided sensitive sequence determination and impurity analysis based on shifts from exact masses. Spectra from protamine sulfate active pharmaceutical ingredient (API) suppliers and from a formulated drug product purchased from the US market were examined. Based on these marketplace survey data, NMR acceptance criteria for chum salmon derived protamine sulfate could be based on the absence of aromatic amino acid signals and on ratios of Ala βH/Arg δH, Gly αH/Arg δH and Arg αH/Arg δH integrated areas of 2.4 ± 1 %, 9.4 ± 3 % and 50 ± 5 %, respectively. For MS, acceptance criteria based on the presence of specific mass to charge (
m/z
) ratio peaks (m/
z
= +8 of 530.455, 540.841, 532.208 and 508.950) could be used for the four major peptides present in the mixture with relative abundances of 17 ± 1 %, 31 ± 2 %, 27 ± 1 % and 25 ± 3 %, respectively. The specificity of the combined NMR and MS assay was tested by comparison to data obtained from herring protamine which contains a different mixture of peptides with related amino acid sequences. Both assays were able to clearly distinguish protamine derived from these different natural sources.
Graphical Abstract
NMR and MS provide orthogonal measurements of complex drug characteristics for quality assurance purposes
The application of multiplexed isobaric tandem mass tag (TMT) labeling and an LTQ Orbitrap XL ETD (electron transfer dissociation) hybrid mass spectrometer as a direct approach for qualitative and ...quantitative characterization of glycoproteins is reported. Bovine fetuin was used as a model glycoprotein in this study. For online liquid chromatography–mass spectrometry (LC–MS) analysis, high-resolution, mass accurate full scan MS spectra were acquired in the Orbitrap mass analyzer followed by data-dependent tandem mass spectrometry (MS/MS) with alternating collision-induced dissociation (CID), ETD, and higher-energy collisional dissociation (HCD) scans. An additional in-source dissociation scan was used as a highly sensitive and selective detection method for eluting glycosylated peptides. By alternatively using three different dissociation methods, 23 glycoforms from all 5 corresponding glycopeptides were identified from a trypsin digest of bovine fetuin. With ETD, labile glycans were retained without any signs of carbohydrate cleavage with concurrent fragmentation of the peptide backbone. Glycosylation sites were clearly localized from the ETD fragmentation data. Glycan structure elucidation was accomplished using CID. The CID experiments generated fragment ions predominantly from cleavage of glycosidic bonds without breaking the peptide bond. Novel to this method, the TMT labeling protocol was modified and adapted for higher labeling efficiency, and a TriVersa NanoMate was used to reinfuse samples to improve ETD and HCD spectra of glycopeptides. Quantification with TMT was verified based on the HCD spectra from multiple nonglycopeptides and glycopeptides. This method can be used as a qualitative and quantitative technique for direct characterization of glycoproteins and has applicability for detection of counterfeit glycoprotein drug products.
Glatiramer acetate (GA) is a mixture of synthetic copolymers consisting of four amino acids (glutamic acid, lysine, alanine, and tyrosine) with a labeled molecular weight range of 5000 to 9000 Da. GA ...is marketed as Copaxone™ by Teva for the treatment of multiple sclerosis. Here, the agency has evaluated the structure and composition of GA and a commercially available comparator, Copolymer-1. Modern analytical technologies which can characterize these complex mixtures are desirable for analysis of their comparability and structural “sameness.” In the studies herein, a molecular fingerprinting approach is taken using mass-accurate mass spectrometry (MS) analysis, nuclear magnetic resonance (NMR) (1D-
1
H-NMR, 1D-
13
C-NMR, and 2D NMR), and asymmetric field flow fractionation (AFFF) coupled with multi-angle light scattering (MALS) for an in-depth characterization of three lots of the marketplace drug and a formulated sample of the comparator. Statistical analyses were applied to the MS and AFFF–MALS data to assess these methods’ ability to detect analytical differences in the mixtures. The combination of multiple orthogonal measurements by liquid chromatography coupled with MS (LC–MS), AFFF–MALS, and NMR on the same sample set was found to be fit for the intended purpose of distinguishing analytical differences between these complex mixtures of peptide chains.
Implementation of modern analytical techniques, such as intact mass spectrometry, may allow for more detailed quality assessments of protein therapeutics. The complexity of the protein therapeutic ...manufacturing process as well as the sensitivity of these drugs to different storage conditions can lead to the presence of several undesired products, including truncations, degradation products, byproducts, and differentially modified protein variants that are difficult to detect by peptide mapping. Intact mass spectrometry can be used to identify the intact protein composition, inclusive of post-translational modifications (PTMs) but can also generate a chemical fingerprint of the different protein species present in a given sample. In this work, we systematically evaluated the influence of multiple charge states, multiple isotopes per charge state, and operating resolution on the suitability of intact mass spectrometry for quantitative analysis using insulin and somatotropin as model systems. Standard curves could be generated using absolute intensity data or using the relative ratio between the analyte and internal standard. These methods demonstrate the validity of quantitative intact mass spectrometry for the analysis of protein therapeutic drugs, thus providing a foundation for future comparative methods.
Abacavir drug hypersensitivity in HIV-treated patients is associated with HLA-B57:01 expression. To understand the immunochemistry of abacavir drug reactions, we investigated the effects of abacavir ...on HLA-B57:01 epitope-binding in vitro and the quality and quantity of self-peptides presented by HLA-B57:01 from abacavir-treated cells.
An HLA-B57:01-specific epitope-binding assay was developed to test for effects of abacavir, didanosine or flucloxacillin on self-peptide binding. To examine whether abacavir alters the peptide repertoire in HLA-B57:01, a B-cell line secreting soluble human leucocyte antigen (sHLA) was cultured in the presence or absence of abacavir, peptides were eluted from purified human leucocyte antigen (HLA), and the peptide epitopes comparatively mapped by mass spectroscopy to identify drug-unique peptides.
Abacavir, but not didansosine or flucloxacillin, enhanced binding of the FITC-labeled self-peptide LF9 to HLA-B57:01 in a dose-dependent manner. Endogenous peptides isolated from abacavir-treated HLA-B57:01 B cells showed amino acid sequence differences compared with peptides from untreated cells. Novel drug-induced peptides lacked typical carboxyl (C) terminal amino acids characteristic of the HLA-B57:01 peptide motif and instead contained predominantly isoleucine or leucine residues. Drug-induced peptides bind to soluble HLA-B57:01 with high affinity that was not altered by abacavir addition.
Our results support a model of drug-induced autoimmunity in which abacavir alters the quantity and quality of self-peptide loading into HLA-B57:01. Drug-induced loading of novel self-peptides into HLA, possibly by abacavir either altering the binding cleft or modifying the peptide-loading complex, generates an array of neo-antigen peptides that drive polyclonal T-cell autoimmune responses and multiorgan systemic toxicity.
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