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The binding affinity of monoclonal antibodies (mAbs) for their intended therapeutic targets is often affected by chemical and post-translational modifications in the antigen binding (Fab) domains. A new two-dimensional analytical approach is described here utilizing native size exclusion chromatography (SEC) to separate populations of antibodies and bound antibody-antigen complexes for subsequent characterization of these modifications by reversed-phase (RP) liquid chromatography-mass spectrometry (LC-MS) at the intact antibody level. Previously, we utilized peptide mapping to measure modifications impacting binding. However, in this study, the large size of the modification (N-glycosylation) allowed assessing its impact from small amounts (â¼20 ug) of intact antibody, without the need for peptide mapping. Here, we apply the native SEC-based competitive binding assay to quickly and qualitatively investigate the effects of Fab glycosylation of four antispike protein mAbs that were developed for use in the treatment of COVID-19 disease. Three of the mAbs were observed to have consensus N-glycosylation sites (N-X-T/S) in the Fab domains, a relatively rare occurrence in therapeutic mAbs. The goal of the study was to characterize the levels of Fab glycosylation present, as well as determine the impact of glycosylation on binding to the spike protein receptor binding domain (RBD) and the ability of the mAbs to inhibit RBD-ACE2 interaction at the intact antibody level, with minimal sample treatment and preparation. The three mAbs with Fab N-glycans were found to have glycosylation profiles ranging from full occupancy at each Fab (in one mAb) to partially glycosylated with mixed populations of two, one, or no glycan moieties. Competitive SEC analysis of mAb-RBD revealed that the glycosylated antibody populations outcompete their nonglycosylated counterparts for the available RBD molecules. This competitive SEC binding analysis was applied to investigate the three-body interaction of a glycosylated mAb blocking the interaction between endogenous binding partners RBD-ACE2, finding that both glycosylated and nonglycosylated mAb populations bound to RBD with high enough affinity to block RBD-ACE2 binding.
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COVID-19 , Humanos , Glicosilación , Cromatografía Liquida , Enzima Convertidora de Angiotensina 2/metabolismo , Espectrometría de Masas en Tándem , Anticuerpos Antivirales , Unión Proteica , Cromatografía en GelRESUMEN
Antibodies facilitate targeted cell killing by engaging with immune cells such as natural killer cells through weak binding interactions with Fcγ receptors on the cell surface. Here, we evaluate the binding affinity of the receptor FcγRIIIa V158 (CD16a) for several therapeutic antibody classes, isoforms, and Fc-fusion proteins using an immobilized receptor affinity liquid chromatography (LC) approach coupled with online mass spectrometry (MS) detection. Aglycosylated FcγRIIIa was used in the affinity chromatography and compared with published affinities using glycosylated receptors. Affinity LC-MS differentiated the IgG1 antibodies primarily according to their Fc glycosylation patterns, with highly galactosylated species having greater affinity for the immobilized receptors and thus eluting later from the column (M5< G0F < G0 afucosylated â G1F < G2F). Sialylated species bound weaker to their asialylated counterparts as reported previously. High mannose glycoforms bound weaker than G0F, contrary to previously published studies using glycosylated receptors. Also, increased receptor binding affinity associated with afucosylated antibodies was not observed with the aglycosylated FcγRIIIa. This apparent difference from previous findings highlighted the importance of the glycans on the receptors for mediating stronger binding interactions. Characterization of temperature-stressed samples by LC-MS peptide mapping revealed over 200 chemical and post-translational modifications, but only the Fc glycans, deamidation of EU N325, and an unknown modification to either proline or cysteine residues of the hinge region were found to have a statistically significant impact on binding.Abbreviations: Antibody-dependent cell-mediated cytotoxicity (ADCC), chimeric antigen receptor (CAR), Chinese hamster ovary (CHO), dithiothreitol (DTT), electrospray ionization (ESI), hydrogen-deuterium exchange (HDX), filter aided-sample preparation (FASP), Fcγ receptor (FcγR), fragment crystallizable (Fc), high-pressure liquid chromatography (HPLC), immunoglobulin G (IgG), liquid chromatography (LC), monoclonal antibody (mAb), mass spectrometry (MS), natural killer (NK), N-glycolylneuraminic acid (NGNA), N-acetylneuraminic acid (NANA), principal component analysis (PCA), surface plasmon resonance (SPR), trifluoroacetic acid (TFA), and extracted mass chromatogram (XMC).
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Cromatografía de Afinidad , Fragmentos Fc de Inmunoglobulinas/química , Espectrometría de Masas , Receptores de IgG/química , Proteínas Recombinantes de Fusión/química , Animales , Células CHO , Cricetulus , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/inmunología , Receptores de IgG/genética , Receptores de IgG/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Rapid technological advances have significantly improved the capability, versatility, and robustness of mass spectrometers which has led to them playing a central role in the development, characterization, and regulatory filings of biopharmaceuticals. Their application spans the entire continuum of drug development, starting with discovery research through product development, characterization, and marketing authorization and continues well into product life cycle management. The scope of application extends beyond traditional protein characterization and includes elements like clone selection, cell culture physiology and bioprocess optimization, investigation support, and process analytical technology. More recently, advances in the MS-based multi-attribute method are enabling the introduction of MS in a cGMP environment for routine release and stability testing. While most applications of MS to date have been for monoclonal antibodies, the successes and learnings should translate to the characterization of next-gen biotherapeutics where modalities like multispecifics could be more prevalent. In this review, we describe the most significant advances in MS and correlate them to the broad spectrum of applications to biotherapeutic development. We anticipate rapid technological improvements to continue that will further accelerate widespread deployment of MS, thereby elevating our overall understanding of product quality and enabling attribute-focused product development.
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Productos Biológicos , Anticuerpos Monoclonales/uso terapéutico , Productos Biológicos/uso terapéutico , Espectrometría de MasasRESUMEN
Therapeutic proteins including antibodies and Fc-fusion proteins undergo a large number of chemical modifications during cell culture, purification, storage and in human circulation. They are also exposed to harsh conditions during stress studies, including elevated temperature, extremes of pH, forced oxidation, physiological pH, UV light to assess the possible degradation pathways and suitability of methods for detecting them. Some of these modifications are located on residues in binding regions, leading to loss of binding and potency and classified as critical quality attributes. Currently, criticality of modifications is assessed by a laborious process of collecting antibody fractions from the soft chromatography techniques ion exchange and hydrophobic interaction chromatography and characterizing the fractions one-by-one for potency and chemical modifications. Here, we describe a method for large-scale, parallel identification of all critical chemical modifications in one experiment. In the first step, the antibody is stressed by one or several stress methods. It is then mixed with target protein and separated by size-exclusion chromatography (SEC) on bound antibody-target complex and unbound antibody. Peptide mapping of fractions and statistical analysis are performed to identify modifications on amino acid residues that affect binding. To identify the modifications leading to slight decreases in binding, competitive SEC of antibody and antigen mixtures was developed and described in a companion study by Shi et al, where target protein is provided at lower level, below the stoichiometry. The newly described method was successfully correlated to crystallography for assessing criticality of chemical modifications and paratope mapping. It is more sensitive to low-level modifications, better streamlined and platform ready.
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Anticuerpos Monoclonales/metabolismo , Complejo Antígeno-Anticuerpo , Antígenos/metabolismo , Cromatografía en Gel , Mapeo Epitopo , Epítopos , Inmunoglobulina G/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Antígenos/inmunología , Sitios de Unión de Anticuerpos , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Mapeo Peptídico , Estabilidad Proteica , Relación Estructura-ActividadRESUMEN
Chemical modifications (attributes) in the binding regions of stressed therapeutic proteins may affect binding to target and efficacy of therapeutic proteins. The method presented here describes the criticality assessment of therapeutic antibody modifications by size-exclusion chromatography (SEC) of competitive binding between a stressed antibody and its target, human epidermal growth factor receptor-2 (HER2), followed by SEC fractionation and peptide mapping characterization of bound and unbound antibodies. When stressed antibody and its target were mixed at a stoichiometric molar ratio of 1:2, only antibody-receptor complex eluted from SEC, indicating that binding was not decreased to break the complex. When a smaller amount of the receptor was provided (1:1), the antibody species with modifications reducing binding eluted as unbound from SEC, while the antibody-receptor complex eluted as the bound fraction. Peptide mapping revealed ratios of modifications between unbound and bound fractions. Statistical analysis after triplicate measurements (n = 3) indicated that heavy chain (HC) D102 isomerization and light chain (LC) N30 deamidation were four-fold higher in unbound fraction with high statistical significance. Although HC N55 deamidation and M107 oxidation were also abundant, they were not statistically different between unbound and bound. Our findings agree with previously published potency measurements of collected CEX fractions and the crystal structure of antibody and HER2. Overall, competitive SEC of stressed antibody-receptor mixture followed by peptide mapping is a useful tool in revealing critical residues and modifications involved in the antibody-target binding, even if they elute as a complex from SEC when mixed at 1:2 stoichiometric ratio.
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Antígenos/metabolismo , Cromatografía en Gel , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/metabolismo , Receptor ErbB-2/metabolismo , Trastuzumab/metabolismo , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Antígenos/química , Antígenos/inmunología , Unión Competitiva , Cromatografía Líquida de Alta Presión , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Luz , Unión Proteica , Estabilidad Proteica , Receptor ErbB-2/química , Receptor ErbB-2/inmunología , Dispersión de Radiación , Espectrofotometría Ultravioleta , Relación Estructura-Actividad , Espectrometría de Masas en Tándem , Trastuzumab/química , Trastuzumab/inmunologíaRESUMEN
Recently, cation exchange chromatography (CEX) using aqueous volatile buffers was directly coupled with mass spectrometry (MS) and applied for intact analysis of therapeutic proteins and antibodies. In our study, chemical modifications responsible for charge variants were identified by CEX-UV-MS for a monoclonal antibody (mAb), a bispecific antibody, and an Fc-fusion protein. We also report post-CEX column addition of organic solvent and acid followed by mixing at elevated temperatures, which unfolded proteins, increased ion intensity (sensitivity) and facilitated top-down analysis. mAb stressed by hydrogen peroxide oxidation was used as a model system, which produced additional CEX peaks. The on-line CEX-UV-MS top-down analysis produced gas-phase fragments containing one or two methionine residues. Oxidation of some methionine residues contributed to earlier (acidic), some to later (basic) eluting peaks, while oxidation of other residues did not change CEX elution. The abundance of the oxidized and non-oxidized fragment ions also allowed estimation of the oxidation percentage of different methionine residues in stressed mAb. CEX-UV-MS measurement revealed a new intact antibody proteoform at 5% that eluted as a basic peak and included paired modifications: high-mannose glycosylation and remaining C-terminal lysine residue (M5/M5 + K). This finding was confirmed by peptide mapping and on-column disulfide reduction coupled with reversed-phase liquid chromatography - top-down MS analysis of the collected basic peak. Overall, our results demonstrate the utility of the on-line method in providing site-specific structural information of charge modifications without fraction collection and laborious peptide mapping.
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Anticuerpos Biespecíficos/análisis , Anticuerpos Monoclonales/análisis , Cromatografía por Intercambio Iónico/métodos , Fragmentos de Inmunoglobulinas/análisis , Espectrometría de Masas/métodos , Animales , Humanos , Mapeo Peptídico/métodosRESUMEN
The Russian Mass Spectrometry Interest Group (RMSIG) emerged in 1998 during the annual ASMS meeting in Orlando, FL. The original goal of the group was to help assimilating mass spectrometrists from the former Soviet Union countries into the West. Following the fulfillment of this objective, the RMSIG continues nowadays as a social and scientific club of 200+ members, to the benefit of mass spectrometry at large. Herein, we share with you the tale of the RMSIG: its history, accomplishments, and present days activities-all in a close relation to ASMS.
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PURPOSE: To show and rationalize the confounding effects on the rotational/oscillatory rheology of surface active impurities in commercial protein formulations such as bovine serum albumin, BSA. METHODS: Bulk and interfacial rotational/oscillatory rheology were used to study the viscosity, complex viscosity, storage/elastic modulus, G' and loss/viscous modulus, G", as a function of time of aqueous formulations of BSA and their purified components. RESULTS: Viscosity/time profiles at steady shear for different commercial BSA products and lots showed viscosity increase, decrease and time-independent profiles at low shear rates. All lots showed shear thinning. BSA monomer and dimers/aggregates, in general, showed similar profiles. Addition of low levels of surfactant or high shear rates rendered all solutions to be Newtonian-like. Interfacial viscosity studies paralleled those on the rotational rheometer. G' > G" with viscosity increase and G' < G" with viscosity decrease over time. CONCLUSIONS: We provide a rational explanation for the time-dependent and source-dependent rheological behavior of aqueous formulations of commercially available BSA proteins based on the migration of protein and surface active impurities to the air/water interface within the rheometer plates leading to the formation and breakdown of protein networks. Highly purified proteins is warranted in rheological studies of protein drug product candidates.
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Albúmina Sérica Bovina/química , Animales , Bovinos , Composición de Medicamentos , Módulo de Elasticidad , Agregado de Proteínas , Estabilidad Proteica , Reología/métodos , Viscosidad , Agua/químicaRESUMEN
Mass spectrometry (MS)-based methods for analyzing protein higher order structures have gained increasing application in the field of biopharmaceutical development. The predominant methods used in this area include native MS, hydrogen deuterium exchange-MS, covalent labeling, cross-linking and limited proteolysis. These MS-based methods will be briefly described in this article, followed by a discussion on how these methods contribute at different stages of discovery and development of protein therapeutics.
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Medición de Intercambio de Deuterio/métodos , Desarrollo de Medicamentos/métodos , Espectrometría de Masas/métodos , Proteínas Recombinantes/metabolismo , Animales , Productos Biológicos/química , Productos Biológicos/metabolismo , Humanos , Conformación Proteica , Proteínas Recombinantes/químicaRESUMEN
Heat-induced conformational transitions are frequently used to probe the free energy landscapes of proteins. However, the extraction of information from thermal denaturation profiles pertaining to non-native protein conformations remains challenging due to their transient nature and significant conformational heterogeneity. Previously we developed a temperature-controlled electrospray ionization (ESI) source that allowed unfolding and association of biopolymers to be monitored by mass spectrometry (MS) in real time as a function of temperature. The scope of this technique is now extended to systems that undergo multi-step denaturation upon heat stress, as well as relatively small-scale conformational changes that are precursors to protein aggregation. The behavior of two therapeutic proteins (human antithrombin and an IgG1 monoclonal antibody) under heat-stress conditions is monitored in real time, providing evidence that relatively small-scale conformational changes in each system lead to protein oligomerization, followed by aggregation. Temperature-controlled ESI MS is particularly useful for the studies of heat-stressed multi-domain proteins such as IgG, where it allows distinct transitions to be observed. The ability of native temperature-controlled ESI MS to monitor both the conformational changes and oligomerization/degradation with high selectivity complements the classic calorimetric methods, lending itself as a powerful experimental tool for the thermostability studies of protein therapeutics.
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Calor , Conformación Proteica , Proteínas/química , Espectrometría de Masa por Ionización de Electrospray , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/uso terapéutico , Antitrombinas/química , Antitrombinas/uso terapéutico , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/uso terapéutico , Desnaturalización Proteica , Estabilidad Proteica , Proteínas/uso terapéuticoRESUMEN
The authors present a clinical case of a 44-year-old male patient with a chronic giant aortic arch pseudoaneurysm with a diameter of 136 × 72 mm. The open resection of false aneurysm was accomplished without artificial circulation. Repair was performed with temporary ascending-to-descending and brachiocephalic bypass without cardiopulmonary bypass.
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Bio-based succinic acid production can redirect industrial chemistry processes from using limited hydrocarbons to renewable carbohydrates. A fermentation process that does not require pH-titrating agents will be advantageous to the industry. Previously, a Yarrowia lipolytica strain that was defective for succinate dehydrogenase was constructed and was found to accumulate up to 17.5 g L(-1) of succinic acid when grown on glycerol without buffering. Here, a derivative mutant was isolated that produced 40.5 g L(-1) of succinic acid in 36 h with a yield of 0.32 g g(-1) glycerol. A combination approach of induced mutagenesis and metabolic evolution allowed isolation of another derivative that could utilize glucose efficiently and accumulated 50.2 g L(-1) succinic acid in 54 h with a yield of 0.43 g g(-1) . The parent strain of these isolated mutants was used for [1,6-(13) C2 ]glucose assimilation analysis. At least 35% glucose was estimated to be utilized through the pentose phosphate pathway, while ≥84% succinic acid was formed through the oxidative branch of the tricarboxylic acid cycle. Biotechnol. Bioeng. 2016;113: 2425-2432. © 2016 Wiley Periodicals, Inc.
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Isótopos de Carbono/farmacocinética , Análisis de Flujos Metabólicos/métodos , Succinato Deshidrogenasa/metabolismo , Ácido Succínico/metabolismo , Yarrowia/fisiología , Glucosa/metabolismo , Tasa de Depuración Metabólica , Succinato Deshidrogenasa/deficiencia , Succinato Deshidrogenasa/genéticaRESUMEN
Monitoring amino acids and vitamins is important for understanding human health, food nutrition and the culture of mammalian cells used to produce therapeutic proteins in biotechnology. A method including ion pairing reversed-phase liquid chromatography with tandem mass spectrometry was developed and optimized to quantify 21 amino acids and 9 water-soluble vitamins in Chinese hamster ovary (CHO) cells and culture media. By optimizing the chromatographic separation, scan time, monitoring time window, and sample preparation procedure, and using isotopically labeled (13)C, (15)N and (2)H internal standards, low limits of quantitation (≤0.054 mg/L), good precision (<10%) and good accuracy (100±10%) were achieved for nearly all the 30 compounds. Applying this method to CHO cell extracts, statistically significant differences in the metabolite levels were measured between two cell lines originated from the same host, indicating differences in genetic makeup or metabolic activities and nutrient supply levels in the culture media. In a fed-batch process of manufacturing scale bioreactors, two distinguished trends for changes in amino acid concentrations were identified in response to feeding. Ten essential amino acids showed a zigzag pattern with maxima at the feeding days, and 9 non-essential amino acids displayed a smoothly changing profile as they were mainly products of cellular metabolism. Five of 9 vitamins accumulated continuously during the culture period, suggesting that they were fed in access. The method serves as an effective tool for the development and optimization of mammalian cell cultures.
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Aminoácidos/análisis , Medios de Cultivo/análisis , Espectrometría de Masas en Tándem/métodos , Vitaminas/análisis , Animales , Células CHO , Cromatografía Liquida/métodos , Cricetinae , Cricetulus , HumanosRESUMEN
Both recombinant and natural human IgG2 antibodies have several different disulfide bond isoforms, which possess different global structures, thermal stabilities, and biological activities. A detailed mapping of the structural difference among IgG2 disulfide isoforms, however, has not been established. In this work, we employed hydrogen/deuterium exchange mass spectrometry to study the conformation of three major IgG2 disulfide isoforms known as IgG2-B, IgG2-A1, and IgG2-A2 in two recombinant human IgG2 monoclonal antibodies. By comparing the protection factors between amino acid residues in isoforms B and A1 (the classical form), we successfully identified several local regions in which the IgG2-B isoform showed more solvent protection than the IgG2-A1 isoform. On the basis of three-dimensional structural models of IgG2, these identified regions were located on the Fab domains, close to the hinge, centered on the side where the two Fab arms faced each other in spatial proximity. We speculated that in the more solvent-protected B isoform, the two Fab arms were brought into contact by the nonclassical disulfide bonds, resulting in a more compact global structure. Loss of Fab domain flexibility in IgG2-B could limit its ability to access cell-surface epitopes, leading to reduced antigen binding potency. The A2 isoform was previously found to have disulfide linkages similar to those of the classical A1 isoform, but with different biophysical behaviors. Our data indicated that, compared to IgG2-A1, IgG2-A2 had less solvent protection in some heavy-chain Fab regions close the hinge, suggesting that the A2 isoform had more flexible Fab domains.
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Anticuerpos Monoclonales/química , Disulfuros/química , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/química , Medición de Intercambio de Deuterio/métodos , Humanos , Espectrometría de Masas/métodos , Estructura Cuaternaria de ProteínaRESUMEN
High mannose (HM) glycan levels on secreted monoclonal antibodies can be influenced by external factors, including osmolality and copper deficiency, and by intrinsic factors determined by different cell lines. In order to identify the metabolic markers associated with HM glycan levels, metabolomics analysis was performed to assess the changes in the extracellular metabolites of recombinant cell lines at different time points during fed-batch production process. Ornithine was identified as the common metabolic marker influenced by both external and intrinsic factors when eight different medium conditions and eight different cell lines exhibiting different levels of HM were compared. A strong correlation was also observed between HM and mRNA expression levels of arginase 1, an enzyme that catalyzes the conversion of arginine to ornithine. The results from functional validation study showed that the supplementation of ornithine to the culture medium leads to an increased level of HM, while reduced concentration of spermine, a downstream product of ornithine metabolism, leads to a decreased level of HM. Additional metabolic markers correlating with HM glycan levels were identified from eight-cell line comparison analysis. A common feature shared by these identified markers is their previously described roles as contributors of cellular redox regulation.
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Anticuerpos Monoclonales/metabolismo , Manosa/metabolismo , Polisacáridos/metabolismo , Animales , Arginasa/genética , Células CHO , Cobre/metabolismo , Cricetinae , Cricetulus , Medios de Cultivo , Metabolómica , Ornitina/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Espermina/metabolismoRESUMEN
Soy hydrolysates are widely used as a nutrient supplement in mammalian cell culture for the production of recombinant proteins. The batch-to-batch variability of a soy hydrolysate often leads to productivity differences. This report describes our metabolomics platform, which includes a battery of LC-MS/MS modes of operation, and advanced data analysis software for automated data processing. The platform was successfully used for screening productivity markers in soy hydrolysates during the production of two therapeutic antibodies in two Chinese hamster ovary cell lines. A total of 123 soy hydrolysate batches were analyzed, from which 62 batches were used in the production runs of cell line #1 and 12 batches were used in the production runs of cell line #2. For cell line #1, out of 19 amino acids, 106 other metabolites and 4,131 peptides identified in the soy hydrolysate batches being used, several nucleosides and short hydrophobic peptides showed negative correlation with antibody titer, while ornithine, citrulline and several amino acids and organic acids correlated positively with titer. For cell line #2, only ornithine and citrulline showed strong positive correlation. When ornithine was spiked into the culture media, both cell lines demonstrated accelerated cell growth, indicating ornithine as a root cause of the performance difference. It is proposed that better soy hydrolysate performance resulted from better bacterial fermentation during the hydrolysate production. A few selected markers were used to predict the performance of other soy hydrolysate batches for cell line #1. The predicted titers agreed with the experimental values with good accuracy.
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Biomarcadores/análisis , Reactores Biológicos , Metaboloma/fisiología , Metabolómica/métodos , Hidrolisados de Proteína/análisis , Proteínas de Soja/análisis , Animales , Biomarcadores/metabolismo , Células CHO , Supervivencia Celular , Cricetinae , Cricetulus , Espectrometría de Masas , Análisis de Componente Principal , Hidrolisados de Proteína/metabolismo , Proteínas Recombinantes , Proteínas de Soja/metabolismoRESUMEN
Ethylene oxide (EtO) is widely used in sterilization of drug product primary containers and medical devices. The impact of residual EtO on protein therapeutics is of significant interest in the biopharmaceutical industry. The potential for EtO to modify individual amino acids in proteins has been previously reported. However, specific identification of EtO adducts in proteins and the effect of residual EtO on the stability of therapeutic proteins has not been reported to date. This paper describes studies of residual EtO with two therapeutic proteins, a PEGylated form of the recombinant human granulocyte colony-stimulating factor (Peg-GCSF) and recombinant human erythropoietin (EPO) formulated with human serum albumin (HSA). Peg-GCSF was filled in an EtO sterilized delivery device and incubated at accelerated stress conditions. Glu-C peptide mapping and LC-MS analyses revealed residual EtO reacted with Peg-GCSF and resulted in EtO modifications at two methionine residues (Met-127 and Met-138). In addition, tryptic peptide mapping and LC-MS analyses revealed residual EtO in plastic vials reacted with HSA in EPO formulation at Met-328 and Cys-34. This paper details the work conducted to understand the effects of residual EtO on the chemical stability of protein therapeutics.
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Eritropoyetina/química , Óxido de Etileno/química , Factor Estimulante de Colonias de Granulocitos/química , Albúmina Sérica/química , Secuencia de Aminoácidos , Eritropoyetina/genética , Factor Estimulante de Colonias de Granulocitos/genética , Humanos , Datos de Secuencia Molecular , Preparaciones Farmacéuticas/química , Albúmina Sérica/genéticaRESUMEN
Insufficient copper level in the mammalian cell culture medium resulted in lactate accumulation while maintaining similar growth and culture viability profiles. Label-free, LC-MS/MS-based shotgun proteomics method was applied to compare the protein expression profiles obtained from the cultures exposed to suboptimal copper level to those provided with sufficient amount of copper. Under copper deficient condition, a substantial reduction of the protein levels of the multiple subunits of Complex IV, also known as cytochrome c oxidase, of the mitochondrial electron transport chain was observed for all three different Chinese Hamster Ovary (CHO) cell lines expressing therapeutic monoclonal antibodies tested. Additional proteins affected by suboptimal copper level included peroxiredoxin (PRDX) and hepatocyte-derived growth factor (HDGF), which were affected during early phase of the fed-batch production, several days prior to initiation of lactate accumulation. In contrast, proteins such as syntenin (SDCBP) and integral membrane 2C (ITM2C) showed altered expression patterns toward the end of culture duration, after lactate divergence had occurred. For all conditions tested, time was the most predominant factor facilitating the direction of global protein expression trend, with substantial number of proteins subjected to time-dependent changes in expression, independent of copper.
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Cobre/deficiencia , Cobre/metabolismo , Proteómica/métodos , Animales , Células CHO , Cricetinae , Cricetulus , Complejo IV de Transporte de Electrones/metabolismo , Ácido Láctico/metabolismoRESUMEN
Quality control operates at different steps in translation to limit errors to approximately one mistranslated codon per 10,000 codons during mRNA-directed protein synthesis. Recent studies have suggested that error rates may actually vary considerably during translation under different growth conditions. Here we examined the misincorporation of Phe at Tyr codons during synthesis of a recombinant antibody produced in tyrosine-limited Chinese hamster ovary (CHO) cells. Tyr to Phe replacements were previously found to occur throughout the antibody at a rate of up to 0.7% irrespective of the identity or context of the Tyr codon translated. Despite this comparatively high mistranslation rate, no significant change in cellular viability was observed. Monitoring of Phe and Tyr levels revealed that changes in error rates correlated with changes in amino acid pools, suggesting that mischarging of tRNA(Tyr) with noncognate Phe by tyrosyl-tRNA synthetase was responsible for mistranslation. Steady-state kinetic analyses of CHO cytoplasmic tyrosyl-tRNA synthetase revealed a 25-fold lower specificity for Tyr over Phe as compared with previously characterized bacterial enzymes, consistent with the observed increase in translation error rates during tyrosine limitation. Functional comparisons of mammalian and bacterial tyrosyl-tRNA synthetase revealed key differences at residues responsible for amino acid recognition, highlighting differences in evolutionary constraints for translation quality control.
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Sustitución de Aminoácidos , Codón , Biosíntesis de Proteínas , Tirosina-ARNt Ligasa/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Tirosina-ARNt Ligasa/genéticaRESUMEN
Mammalian cell culture performance is influenced by both intrinsic (genetic) and extrinsic (media and process) factors. In this study, intrinsic capacity of various monoclonal antibody-producing Chinese Hamster Ovary (CHO) cell lines was compared by exposing them to the same culture condition. Microarray-based transcriptomics and LC-MS/MS shotgun proteomics technologies were utilized to obtain expression landscape of different cell lines. Specific transcripts and proteins correlating with productivity, growth rate and cell size have been identified. The proteomics analysis results showed a strong correlation between the intracellular protein expression levels of the recombinant DHFR and productivity. In contrast, neither the light chain nor the heavy chain of the recombinant monoclonal antibody showed correlation to productivity. Other top ranked proteins which demonstrated positive correlation to productivity included the adaptor protein complex subunits AP3D1and AP2B2, DNA repair protein DDB1 and the ER translocation complex component, SRPR. The subunits of molecular chaperone T-complex protein 1 and the regulator of mitochondrial one-carbon metabolism MTHFD2 showed negative correlation to productivity. The transcriptomics analysis has identified the regulators of calcium signaling, Tmem20 and Rcan1, as the top ranked genes displaying positive and negative correlation to productivity, respectively. For the second part of the study, the principal component analysis (PCA) was generated to view the underlying global structure of the expression data. A clear division and expression polarity was observed between the two distinct clusters of cell lines, independent of link to productivity or any other traits examined. The primary component of the PCA generated from either transcriptomics or proteomics data displayed a strong correlation to cell size and doubling time, while none of the main principal components showed correlation to productivity. Our findings suggest that productivity is rather a minor feature in the context of global transcriptional or protein expression space.