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1.
Anal Bioanal Chem ; 414(2): 747-758, 2022 Jan.
Article in English | MEDLINE | ID: mdl-34595561

ABSTRACT

During biologics development, manufacturers must demonstrate clearance of host cell impurities and contaminants to ensure drug purity, manufacturing process consistency, and patient safety. Host cell proteins (HCPs) are a major class of process-related impurities and require monitoring and documentation of their presence through development and manufacturing. Even in residual amounts, they are known to affect product quality and efficacy as well as patient safety. HCP analysis using enzyme-linked immunosorbent assay (HCP-ELISA) is the standard technique, due to its simple handling, short analysis time, and high sensitivity for protein impurities. Liquid chromatography mass spectrometry (LC-MS) is an orthogonal method for HCP analysis and is increasingly included in regulatory documentation. LC-MS offers advantages where HCP-ELISA has drawbacks, e.g., the ability to identify and quantify individual HCPs. This article summarizes the available knowledge about monitoring HCPs in biologics and presents the newest trends in HCP analysis with current state-of-the-art HCP measurement tools. Through case studies, we present examples of HCP control strategies that have been used in regulatory license applications, using an MS-based coverage analysis and HCP-ELISA and LC-MS for HCP quantification. This provides novel insight into the rapid evolving strategy of HCP analysis. Improvements in technologies to evaluate HCP-ELISA suitability and the implementation of orthogonal LC-MS methods for HCP analysis are important to rationally manipulate, engineer, and select suitable cell lines and downstream processing steps to limit problematic HCPs.


Subject(s)
Biological Products/metabolism , Chromatography, Liquid/methods , Enzyme-Linked Immunosorbent Assay/methods , Mass Spectrometry/methods , Proteins/metabolism , Animals , Cell Line
2.
Biotechnol Prog ; 36(4): e2983, 2020 07.
Article in English | MEDLINE | ID: mdl-32087048

ABSTRACT

Monitoring host cell proteins (HCPs) is one of the most important analytical requirements in production of recombinant biopharmaceuticals to ensure product purity and patient safety. Enzyme-linked immunosorbent assay (ELISA) is the standard method for monitoring HCP clearance. It is important to validate that the critical reagent of an ELISA, the HCP antibody, covers a broad spectrum of the HCPs potentially present in the purified drug substance. Current coverage methods for assessing HCP antibody coverage are based on 2D-Western blot or immunoaffinity-purification combined with 2D gel electrophoresis and have several limitations. In the present study, we present a novel coverage method combining ELISA-based immunocapture with protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS): ELISA-MS. ELISA-MS is used to accurately determine HCP coverage of an early process sample by three commercially available anti-Escherichia coli HCP antibodies, evading the limitations of current methods for coverage analysis, and taking advantage of the benefits of MS analysis. The results obtained comprise a list of individual HCPs covered by each HCP antibody. The novel method shows high sensitivity, high reproducibility, and enables tight control of nonspecific binding through inclusion of a species-specific isotype control antibody. We propose that ELISA-MS will be a valuable supplement to existing coverage methods or even a replacement. ELISA-MS will increase the possibility of selecting the best HCP ELISA, thus improving HCP surveillance and resulting in a final HCP profile with the lowest achievable risk. Overall, this will be beneficial to both the pharmaceutical industry and patient safety.


Subject(s)
Antibodies, Monoclonal/isolation & purification , Enzyme-Linked Immunosorbent Assay/methods , Proteins/isolation & purification , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , CHO Cells/immunology , Chromatography, Liquid/methods , Cricetinae , Cricetulus , Humans , Proteins/immunology , Species Specificity , Tandem Mass Spectrometry/methods
3.
Allergy ; 75(7): 1721-1729, 2020 07.
Article in English | MEDLINE | ID: mdl-31715004

ABSTRACT

BACKGROUND: There is mounting evidence that systemic uptake of food allergens is key to triggering anaphylaxis. However, direct proof for this theory is still lacking. The purpose of this study was to quantify the absorption and to determine the absorption kinetics of immunoreactive peanut protein in relation to the allergic response in human. METHODS: Quantitative protein assays including mass spectrometry, dot blots and Western blotting were developed to determine the level of Ara h 2 absorption in human serum. The double monoclonal sandwich ELISA was applied to quantify absorbed Ara h 2 and 6, and the basophil histamine release assay and the human passive cutaneous anaphylaxis test were utilized to study the absorption kinetics of immunologically intact peanut proteins. RESULTS: The protein assays worked but were not sensitive enough to trace the minute amounts of absorbed Ara h 2 in human serum. The level of Ara h 6 in serum was found to be up to 0.2 ng/mL, but Ara h 2 could not be detected with the ELISA. Both the in vivo and the in vitro methods were successful in demonstrating that: immunoreactive peanut protein was absorbed shortly after ingestion (≤5 minutes); the peanut protein concentration peaks between 1 and 4 hours; and peanut proteins can circulate for at least 48 hours in the bloodstream. CONCLUSION: Ingested peanut protein is absorbed systemically and retains its immunoreactive capacity in human serum. However, the precise quantities and the implication for the elicitation of anaphylaxis remains to be elucidated.


Subject(s)
Arachis , Peanut Hypersensitivity , 2S Albumins, Plant , Allergens , Antigens, Plant , Humans , Plant Proteins
4.
Anal Chem ; 90(15): 9055-9059, 2018 08 07.
Article in English | MEDLINE | ID: mdl-30015478

ABSTRACT

Protein sequencing by mass spectrometry has transformed the field of biopharmaceutical analysis, but a missing part in the analytical toolkit is the ability to distinguish between the isomeric residues isoleucine and leucine because it is a requisite for efficient analysis of the primary structure of proteins. To address this need, we have developed a novel mass spectrometric method that combines reductive dimethylation and MS3 fragmentation with LCMS peptide mapping. The dimethylation of peptide N-termini leads to intense a1-ions upon collision-induced fragmentation, and further fragmentation of the isoleucine/leucine a1-ion leads to informative spectra with fragments that can discriminate between the two isomers. The methodology of a1-directed MS3 was applied to two antibodies in combination with the proteases trypsin, thermolysin, chymotrypsin, and pepsin to generate peptides exposing N-terminal I/L residues.

5.
Protein Expr Purif ; 147: 69-77, 2018 07.
Article in English | MEDLINE | ID: mdl-29526817

ABSTRACT

Recombinantly expressed biopharmaceutical proteins often undergo a series of purification steps with the aim of removing contaminating material. Depending on the application of the protein, there are various requirements for the degree of purity, but host cell proteins (HCPs) will in general remain in small amounts. LC-MS has emerged as an orthogonal technique, capable of providing detailed information regarding the individual proteins. The aim of this case study was to characterize the HCPs associated with a biopharmaceutical protein, provided by Statens Serum Institut (DK), which is used in the field of tuberculosis and has not previously been studied by LC-MS. The developed method and acquired experiences served to develop a generalized strategy for HCP-characterization in our laboratory. We evaluated the use of different spectral libraries, recorded in data-dependent mode for obtaining the highest HCP coverage, combined with SWATH-based absolute quantification. The accuracy of two label-free absolute quantification strategies was evaluated using stable isotope peptides. Two different sample preparation workflows were evaluated for optimal HCP yield. . The label-free strategy produced accurate quantification across several orders of magnitude, and the calculated purity was found to be in agreement with previously obtained ELISA data.


Subject(s)
Biological Products/metabolism , Chromatography, Liquid/methods , Peptides/metabolism , Proteins/metabolism , Recombinant Proteins/metabolism , Tandem Mass Spectrometry/methods , Bacteria/genetics , Bacteria/metabolism , Drug Contamination/prevention & control , Humans , Pharmaceutical Preparations/metabolism , Proteins/genetics
6.
Nat Commun ; 5: 3369, 2014 Feb 26.
Article in English | MEDLINE | ID: mdl-24569628

ABSTRACT

Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multidrug-resistant tuberculosis in decades. Though BDQ has shown excellent efficacy in clinical trials, its early bactericidal activity during the first week of chemotherapy is minimal. Here, using microfluidic devices and time-lapse microscopy of Mycobacterium tuberculosis, we confirm the absence of significant bacteriolytic activity during the first 3-4 days of exposure to BDQ. BDQ-induced inhibition of ATP synthesis leads to bacteriostasis within hours after drug addition. Transcriptional and proteomic analyses reveal that M. tuberculosis responds to BDQ by induction of the dormancy regulon and activation of ATP-generating pathways, thereby maintaining bacterial viability during initial drug exposure. BDQ-induced bacterial killing is significantly enhanced when the mycobacteria are grown on non-fermentable energy sources such as lipids (impeding ATP synthesis via glycolysis). Our results show that BDQ exposure triggers a metabolic remodelling in mycobacteria, thereby enabling transient bacterial survival.


Subject(s)
Diarylquinolines/pharmacology , Gene Expression Regulation, Bacterial/drug effects , Glycolysis/drug effects , Mycobacterium tuberculosis/drug effects , Adenosine Triphosphate/metabolism , Antitubercular Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Dose-Response Relationship, Drug , Gene Expression Profiling/methods , Microbial Viability/drug effects , Microbial Viability/genetics , Microfluidic Analytical Techniques , Microscopy, Fluorescence , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Oligonucleotide Array Sequence Analysis , Proteome/genetics , Proteome/metabolism , Proteomics/methods , Reverse Transcriptase Polymerase Chain Reaction , Single-Cell Analysis/methods , Time Factors , Time-Lapse Imaging
7.
Phytopathology ; 102(9): 878-91, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22708726

ABSTRACT

Pathogenic isolates of Pyrenochaeta lycopersici, the causal agent of corky root rot of tomato, secrete cell death in tomato 1 (CDiT1), a homodimeric protein of 35 kDa inducing cell death after infiltration into the leaf apoplast of tomato. CDiT1 was purified by fast protein liquid chromatography, characterized by mass spectrometry and cDNA cloning. Its activity was confirmed after infiltration of an affinity-purified recombinant fusion of the protein with a C-terminal polyhistidine tag. CDiT1 is highly expressed during tomato root infection compared with axenic culture, and has a putative ortholog in other pathogenic Pleosporales species producing proteinaceous toxins that contribute to virulence. Infiltration of CDiT1 into leaves of other plants susceptible to P. lycopersici revealed that the protein affects them differentially. All varieties of cultivated tomato (Solanum lycopersicum) tested were more sensitive to CDiT1 than those of currant tomato (S. pimpinellifolium). Root infection assays showed that varieties of currant tomato are also significantly less prone to intracellular colonization of their root cells by hyphae of P. lycopersici than varieties of cultivated tomato. Therefore, secretion of this novel type of inducer of cell death during penetration of the fungus inside root cells might favor infection of host species that are highly sensitive to this molecule.


Subject(s)
Ascomycota/metabolism , Cucumis/microbiology , Gene Expression Regulation, Fungal/physiology , Plant Diseases/microbiology , Solanaceae/microbiology , Amino Acid Sequence , Ascomycota/physiology , Fungal Proteins/genetics , Fungal Proteins/metabolism , Molecular Sequence Data , Plant Leaves/microbiology , Plant Roots/microbiology
9.
Proteomics ; 9(5): 1230-40, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19253281

ABSTRACT

Members of the transforming growth factor (TGF)-beta superfamily are key regulators of lung development and homeostasis, in particular by controlling alveolar/bronchial epithelial cell function. TGF-beta signaling involves ligand-dependent activation of receptor serine/threonine kinases, activation and subsequent nuclear translocation of pathway-specific transcription factors (Smads), and ultimately, modulation of gene expression. While Smad-dependent responses represent the primary signaling components activated by TGF-beta receptors, their function is controlled by a variety of cofactors. In addition, alternative signaling systems mediating TGF-beta-induced effects have recently been described such as MAP kinase pathways. To uncover novel proteins that participate in TGF-beta signaling via nuclear/cytoplasmic shuttling in lung epithelial cells, we have analyzed A549 human lung epithelial cells, using subcellular fractionation combined with 2-D PAGE, tryptic digestion, and MS. We identified a rapid increase in the cytosolic localization of KH-type splicing regulatory protein (KHSRP), far upstream element-binding protein (FUBP1), hnRNP-L, and hnRNP-H1, concomitant with a decrease in their nuclear localization in response to TGF-beta1. Proteomic data were confirmed by immunofluorescence and immunoblot analyses. In summary, we represent a powerful novel technology for the identification of previously unknown signaling intermediates.


Subject(s)
Epithelial Cells/cytology , Lung/cytology , Proteome/analysis , Subcellular Fractions/chemistry , Subcellular Fractions/metabolism , Transforming Growth Factor beta1/metabolism , Cell Differentiation , Cell Line , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Cytosol/chemistry , Cytosol/metabolism , Electrophoresis, Gel, Two-Dimensional , Epithelial Cells/metabolism , Humans , Lung/metabolism , Proteome/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
10.
Proteomics ; 2(12): 1706-14, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12469340

ABSTRACT

As a consequence of their poor solubility during isoelectric focusing, integral membrane proteins are generally absent from two-dimensional gel proteome maps. In order to analyze the yeast plasma membrane proteome, a plasma membrane purification protocol was optimized in order to reduce contaminating membranes and cytosolic proteins. Specifically, the new fractionation scheme largely depleted the plasma membrane fraction of cytosolic proteins by deoxycholate stripping and ribosomal proteins by sucrose gradient flotation. The plasma membrane complement was resolved by two-dimensional electrophoresis using the cationic detergent cetyl trimethyl ammonium bromide in the first, and sodium dodecyl sulfate in the second dimension, and fifty spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectometry. In spite of the presence of still contaminating ribosomal proteins, major proteins corresponded to known plasma membrane residents, the ABC transporters Pdr5p and Snq2p, the P-type H(+)-ATPase Pma1p, the glucose transporter Hxt7p, the seven transmembrane-span Mrh1p, the low affinity Fe(++) transporter Fet4p, the twelve-span Ptr2p, and the plasma membrane anchored casein kinase Yck2p. The four transmembrane-span proteins Sur7p and Nce102p were also present in the isolated plasma membranes, as well as the unknown protein Ygr266wp that probably contains a single transmembrane span. Thus, combining subcellular fractionation with adapted two-dimensional electrophoresis resulted in the identification of intrinsic plasma membrane proteins.


Subject(s)
Membrane Proteins/chemistry , Proteomics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Cell Fractionation , Cell Membrane/chemistry , Electrophoresis, Gel, Two-Dimensional , Mass Spectrometry , Membrane Proteins/isolation & purification
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