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1.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207579

RESUMO

Biomanufacturing processes may be optimized by storing cell culture media at room temperature, but this is currently limited by their instability and change in color upon long-term storage. This study demonstrates that one of the critical contributing factors toward media browning is tryptophan. LC-MS technology was utilized to identify tryptophan degradation products, which are likely formed primarily from oxidation reactions. Several of the identified compounds were shown to contribute significantly to color in solutions but also to exhibit toxicity against CHO cells. A cell-culture-compatible antioxidant, a-ketoglutaric acid, was found to be an efficient cell culture media additive for stabilizing components against degradation, inhibiting the browning of media formulations, and decreasing ammonia production, thus providing a viable method for developing room-temperature stable cell culture media.


Assuntos
Meios de Cultura/química , Triptofano/metabolismo , Animais , Células CHO , Cricetulus , Oxirredução , Triptofano/análise
2.
Biotechnol Bioeng ; 118(9): 3395-3408, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33738790

RESUMO

Increasing demands for protein-based therapeutics such as monoclonal antibodies, fusion proteins, bispecific molecules, and antibody fragments require researchers to constantly find innovative solutions. To increase yields and decrease costs of next generation bioprocesses, highly concentrated cell culture media formulations are developed but often limited by the low solubility of amino acids such as tyrosine, cystine, leucine, and isoleucine, in particular at physiological pH. This study sought to investigate highly soluble and bioavailable derivatives of leucine and isoleucine that are applicable for fed-batch processes. N-lactoyl-leucine and N-lactoyl-isoleucine sodium salts were tested in cell culture media and proved to be beneficial to increase the overall solubility of cell culture media formulations. These modified amino acids proved to be bioavailable for various Chinese hamster ovary (CHO) cells and were suitable for replacement of canonical amino acids in cell culture feeds. The quality of the final recombinant protein was studied in bioprocesses using the derivatives, and the mechanism of cleavage was investigated in CHO cells. Altogether, both N-lactoyl amino acids represent an advantageous alternative to canonical amino acids to develop highly concentrated cell culture media formulations to support next generation bioprocesses.

3.
Free Radic Biol Med ; 160: 696-718, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-32911085

RESUMO

Tryptophan is one of the essential mammalian amino acids and is thus a required component in human nutrition, animal feeds, and cell culture media. However, this aromatic amino acid is highly susceptible to oxidation and is known to degrade into multiple products during manufacturing, storage, and processing. Many physical and chemical processes contribute to the degradation of this compound, primarily via oxidation or cleavage of the highly reactive indole ring. The central contributing factors are reactive oxygen species, such as singlet oxygen, hydrogen peroxide, and hydroxyl radicals; light and photosensitizers; metals; and heat. In a multi-component mixture, tryptophan also commonly reacts with carbonyl-containing compounds, leading to a wide variety of products. The purpose of this review is to summarize the current state of knowledge regarding the degradation and interaction products of tryptophan in complex liquid solutions and in proteins. For the purposes of context, a brief summary of the key pathways in tryptophan metabolism will be included, along with common methods and issues in tryptophan manufacturing. The review will focus on the conditions that lead to tryptophan degradation, the products generated in these processes, their known biological effects, and methods which may be applied to stabilize the amino acid.


Assuntos
Oxigênio Singlete , Triptofano , Animais , Humanos , Radical Hidroxila , Oxirredução , Espécies Reativas de Oxigênio , Triptofano/metabolismo
4.
Biotechnol Bioeng ; 116(6): 1537-1555, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30793282

RESUMO

Nowadays, chemically defined cell culture media (CCM) have replaced serum- and hydrolysate-based media that rely on complex ingredients, such as yeast extracts or peptones. Benefits include a significantly lower lot-to-lot variability, more efficient manufacturing by reduction to essential components, and the ability to exclude components that may negatively influence growth, viability, or productivity. Even though current chemically defined CCMs provide an excellent basis for various mammalian biotechnological processes, vitamin instabilities are known to be a key factor contributing to the variabilities still present in liquid CCM as well as to short storage times. In this review, the chemical degradation pathways and products for the most relevant vitamins for CCM will be discussed, with a focus on the effects of light, oxygen, heat, and other CCM compounds. Different approaches to stabilize vitamins in solution, such as replacement with analogs, encapsulation, or the addition of stabilizing compounds will also be reviewed. While these vitamins and vitamin stabilization approaches are presented here as particular for CCM, the application of these concepts can also be considered relevant for pharmaceutical, medical, and food supplement purposes. More precise knowledge regarding vitamin instabilities will contribute to stabilize future formulations and thus decrease residual lot-to-lot variability.


Assuntos
Meios de Cultura/química , Vitaminas/química , Animais , Biotecnologia/métodos , Técnicas de Cultura de Células/métodos , Meios de Cultura/metabolismo , Estabilidade de Medicamentos , Excipientes/química , Excipientes/metabolismo , Temperatura Alta , Humanos , Luz , Oxigênio/metabolismo , Vitaminas/metabolismo
5.
MAbs ; 9(6): 889-897, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28581887

RESUMO

The quality of recombinant proteins such as monoclonal antibodies produced using Chinese hamster ovary cell-based mammalian systems is dependent on many factors, including cell line, process and cell culture media. Due to these factors, the generated product is heterogeneous and may have chemically-induced modifications or post-translational modifications that affect antibody stability, functionality and, in some cases, patient safety. This study demonstrates that S-sulfocysteine, a cysteine derivative, can increase the antibody specific productivity in different cell lines cultivated with different processes while minimizing trisulfide linkages in generated mAbs, mainly between heavy and light chain. The supplementation of a cell culture feed with S-sulfocysteine also proved to be useful to reduce the percentage of antibody fragments generated from the monoclonal antibody. Overall, this new component used in the upstream process allows a reduction of product heterogeneity.

6.
Biotechnol Prog ; 33(3): 759-770, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28268250

RESUMO

The ability of cell culture media components to generate reactive species as well as their sensitivity to oxidative degradation, affects the overall stability of media and the behavior of cells cultured in vitro. This study investigates the influence of thiazolidine molecules, formed from the condensation between cysteine and alpha-ketoacids, on the stability of these complex mixtures and on the performance of cell culture processes aiming to produce therapeutically relevant monoclonal antibodies. Results presented in this study indicate that 2-methyl-1,3-thiazolidine-2,4-dicarboxylic acid and 2-(2-carboxyethyl)-1,3-thiazolidine-2,4-dicarboxylic acid, obtained by condensation of cysteine with pyruvate or alpha-ketoglutarate, respectively, are able to stabilize cell culture media formulations, in particular redox sensitive molecules like folic acid, thiamine, l-methionine (met) and l-tryptophan (trp). The use of thiazolidine containing feeds in Chinese hamster ovary fed-batch processes showed prolonged culture duration and increased productivity. This enhanced performance was correlated with lower reactive species generation, extracellularly and intracellularly. Moreover, an anti-oxidative response was triggered via the induction of superoxide dismutase and an increase in the total glutathione pool, the major intracellular antioxidant. In total, the results confirm that cells in vitro are not cultured in an oxidant-free environment, a concept that has to be considered when studying the influence of reactive species in human diseases. Furthermore, this study indicates that thiazolidines are an interesting class of antioxidant molecules, capable of increasing cell culture media stability and process performance. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:759-770, 2017.


Assuntos
Antioxidantes/metabolismo , Tiazolidinas/metabolismo , Animais , Células CHO , Cricetulus , Meios de Cultura , Ácido Fólico/metabolismo , Metionina/metabolismo , Superóxido Dismutase/metabolismo , Tiamina/metabolismo , Triptofano/metabolismo
7.
J Biotechnol ; 218: 53-63, 2016 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-26654938

RESUMO

Industrial fed-batch cultivation of mammalian cells is used for the production of therapeutic proteins such as monoclonal antibodies. Besides medium ensuring initial growth, feeding is necessary to improve growth, viability and antibody production. Established processes include a slight acidic main feed and a separate alkaline feed containing l-tyrosine and l-cysteine. Since l-cysteine is not stable at neutral pH, a new derivative, S-sulfocysteine, was tested in neutral pH feeds. In small scale fed-batch processes, the S-sulfocysteine process yielded a comparable maximum viable cell density, prolonged viability and increased titer compared to the two feed system. Bioreactor experiments confirmed the increase in specific productivity. In depth characterization of the monoclonal antibody indicated no change in the glycosylation, or charge variant pattern whereas peptide mapping experiments were not able to detect any integration of the modified amino acid in the sequence of the monoclonal antibody. Finally, the mechanism of action of S-sulfocysteine was investigated, and results pointed out the anti-oxidative potential of the molecule, mediated through an increase in superoxide dismutase enzyme levels and in the total intracellular glutathione pool. Finally, we propose that the increase in specific productivity obtained in the S-sulfocysteine process results from the anti-oxidative properties of the molecule.


Assuntos
Antioxidantes/farmacologia , Técnicas de Cultura Celular por Lotes/métodos , Meios de Cultura , Cisteína/análogos & derivados , Aminoácidos/metabolismo , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/efeitos dos fármacos , Anticorpos Monoclonais/metabolismo , Antioxidantes/metabolismo , Reatores Biológicos , Células CHO , Cricetinae , Cricetulus , Cisteína/metabolismo , Cisteína/farmacologia , Glutationa/metabolismo , Glicosilação/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Mapeamento de Peptídeos , Tirosina/metabolismo
8.
J Biotechnol ; 186: 110-8, 2014 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-25014403

RESUMO

Fed-batch culture bioprocesses are currently used predominantly for the production of recombinant proteins, especially monoclonal antibodies. In these cultures, concentrated feeds are added during cultivation to prevent nutrient depletion, thus extending the cellular growth phase and increasing product concentrations. One limitation in these bioprocesses arises from the low solubility or stability of some compounds at high concentrations, in particular amino acids. This study describes the synthesis and evaluation of a phosphotyrosine disodium salt as a tyrosine source in fed-batch processes. This molecule is highly soluble in concentrated feeds at neutral pH. Mechanistic studies demonstrated that the molecule is cleaved in the cell culture supernatant after processing by released phosphatases, leading to phosphate and free L-tyrosine which can be taken up by the cells. No intact phosphotyrosine was detected intracellularly or incorporated into the sequence of the monoclonal antibody. The use of this new molecule allows the simplification of fed-batch processes in large scale manufacturing via the implementation of neutral pH, highly concentrated feeds.


Assuntos
Anticorpos Monoclonais/metabolismo , Reatores Biológicos , Meios de Cultura/química , Fosfotirosina/química , Proteínas Recombinantes/metabolismo , Sódio/química , Animais , Anticorpos Monoclonais/química , Células CHO , Cricetinae , Cricetulus , Meios de Cultura/metabolismo , Fosfotirosina/metabolismo , Proteínas Recombinantes/química , Sódio/metabolismo
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