Mechanistic insights into the biological activity of S-Sulfocysteine in CHO cells using a multi-omics approach.

S-Sulfocysteine (SSC), a bioavailable L-cysteine derivative (Cys), is known to be taken up and metabolized in Chinese hamster ovary (CHO) cells used to produce novel therapeutic biological entities. To gain a deeper mechanistic insight into the SSC biological activity and metabolization, a multi-omics study was performed on industrially relevant CHO-K1 GS cells throughout a fed-batch process, including metabolomic and proteomic profiling combined with multivariate data and pathway analyses. Multi-layered data and enzymatical assays revealed an intracellular SSC/glutathione mixed disulfide formation and glutaredoxin-mediated reduction, releasing Cys and sulfur species. Increased Cys availability was directed towards glutathione and taurine synthesis, while other Cys catabolic pathways were likewise affected, indicating that cells strive to maintain Cys homeostasis and cellular functions.

Sodium chloride impacts glycosylation and N- and O-glycan site occupancy of an Fc-fusion protein.

Fc-fusion proteins are highly complex molecules, difficult to manufacture at scale. In this work, undesired proteoforms were detected during the manufacture of a therapeutic fusion protein produced in CHO cells. These species were characterized using gel electrophoresis, size exclusion chromatography and liquid chromatography-mass spectrometry leading to the identification of low molecular weight proteoforms presenting low N- and O-glycan site occupancy, as well as a low sialylation content. Upstream process parameters were investigated, and fusion protein quality was shown to be linked to the sodium chloride content of the medium. A mitigation strategy was developed to avoid formation of unwanted glyco-variants, resulting in an increased yield of highly glycosylated Fc-fusion protein. The effect of sodium chloride was shown to be independent of the osmolality increase and was hypothesized to be linked to a modulation of Golgi acidity, which is required for the correct localization and function of glycosyltransferases. Altogether, this study highlights the importance of the salt balance in cell culture media used to produce highly sialylated and occupied glycoproteins, helping to maximize the yield and increase robustness of processes aiming at producing biopharmaceutical complex therapeutic molecules.

Capsicum baccatum Red Pepper Prevents Cardiometabolic Risk in Rats Fed with an Ultra-Processed Diet.

Metabolic syndrome is a serious health condition reaching epidemic proportions worldwide and is closely linked to an increased risk of cardiovascular problems. The lack of appropriate treatment paves the way for developing new therapeutic agents as a high priority in the current research. In this study, we evaluated the protective effects of Capsicum baccatum red pepper on metabolic syndrome scenarios induced by an ultra-processed diet in rats. After four months, the ultra-processed diet increased central obesity, triglycerides, total cholesterol, LDL-cholesterol plasma levels, and impaired glucose tolerance. The oral administration of C. baccatum concomitantly with the ultra-processed diet avoided the accumulation of adipose tissue in the visceral region, reduced the total cholesterol and LDL fraction, and improved glucose homeostasis, factors commonly associated with metabolic syndrome. The data presented herein reveal an important preventive action of C. baccatum in developing metabolic disorders among animals fed a hypercaloric diet, significantly reducing their cardiometabolic risk. Allied with the absence of toxic effects after chronic use, our study suggests C. baccatum red pepper as a secure and enriched source of bioactive compounds promising to protect against pathological processes associated with metabolic syndrome.

A reflection on the improvement of Chinese hamster ovary cell-based bioprocesses through advances in proteomic techniques.

Chinese hamster ovary (CHO) cells are the preferred mammalian host for the large-scale production of recombinant proteins in the biopharmaceutical industry. Research endeavors have been directed to the optimization of CHO-based bioprocesses to increase protein quantity and quality, often in an empirical manner. To provide a rationale for those achievements, a myriad of CHO proteomic studies has arisen in recent decades. This review gives an overview of significant advances in LC-MS-based proteomics and sheds light on CHO proteomic studies, with a particular focus on CHO cells with superior bioprocessing phenotypes (growth, viability, titer, productivity and cQA), that have exploited novel proteomic or sub-omic techniques. These proteomic findings expand the current knowledge and understanding about the underlying protein clusters, protein regulatory networks and biological pathways governing such phenotypic changes. The proteomic studies, highlighted herein, will help in the targeted modulation of these cell factories to the desired needs.

Nanoemulsion Containing Kojic Dipalmitate and Rosehip Oil: A Promising Formulation to Treat Melasma.

Melasma is a hard-to-treat hyperpigmentation disorder. Combined incorporation of kojic dipalmitate (KDP), the esterified form of kojic acid, and rosehip oil, an oil with antioxidant and skin-regenerating properties, into nanocarrier systems appears to be a suitable strategy to develop high-performance formulations. A high-energy method (Ultra-Turrax®) was used to develop nanoemulsions containing up to 2 mg/mL KDP, 5% rosehip oil, and 7.5% surfactant. Formulations were characterized regarding droplet size, size distribution, pH, density, morphology, KDP content, incorporation efficiency, and stability under different temperature conditions. A scale-up study was conducted. Skin permeation, antioxidant potential, and tyrosinase inhibitory activity were assessed in vitro. Cell viability studies were also performed. Results showed that nanoemulsions containing 1 and 2 mg/mL KDP had incorporation efficiencies greater than 95%, droplet size smaller than 130 nm, suitable size distribution, zeta potential of approximately -10 mV, and good stability over 30 days of refrigerated storage. The nanoemulsion containing 1 mg/mL KDP was chosen for further evaluation because it had lower nanocrystal formation, greater scale-up feasibility and allowed KDP permeation up to the epidermis similarly than observed for 2 mg/mL KDP. This formulation (1 mg/mL KDP) showed antioxidant and depigmenting efficacy, close to that of 1 mM ascorbic acid. No cytotoxicity was observed in formulations concentrations ranging from 0.06% to 1%.

Stability and Requirement for Thiamin in a Cell Culture Feed Used to Produce New Biological Entities.

Thiamin is susceptible to heat and oxidation, which is a concern for the development of concentrated and room temperature stable feeds used to produce recombinant proteins. Hence, it is critical to understand the reactivity and necessity of the vitamin in liquid feeds to be able to either develop mitigation strategies to stabilize the vitamin or to remove thiamin from formulations if it is unnecessary. LC-MS/MS was used to investigate thiamin stability in different liquid feed formulations and to identify thiamin degradation products. Results indicate oxidation of thiamin and interaction with amino acids, keto acids, and sulfur containing components. Thiamin necessity in feed was assessed during a fed batch experiment, focusing on cell performance and critical quality attributes of the produced recombinant proteins. The impact of thiamin depletion in the feed on the intra- and extracellular metabolome was investigated using untargeted LC-MS/MS. Results indicate that thiamin can be removed from the feed without affecting the performance or the intra- and extracellular metabolome of the tested cell lines. Overall, profound insights on thiamin reactivity and necessity are presented in this study, suggesting the removal of the dispensable and instable vitamin as a simple means for the development of next generation feeds used to produce therapeutic biological entities.

Copper impurity of iron raw material contributes to improved cell culture performance.

Cell culture medium (CCM) formulations are chemically defined to reduce lot-to-lot variability and complexity of the medium while still providing all essential nutrients supporting cell growth and productivity of various cell lines. However, raw material impurities may still introduce variations and inconsistencies to final CCM formulations. In one of our previous studies (Weiss et al. Biotechnol Prog. 2021;37(4):e3148), we have demonstrated the impact of iron raw material impurity on Chinese hamster ovary (CHO) cell performance and critical quality attributes (CQAs) of recombinant proteins within the Cellvento® 4CHO CCM platform by identifying manganese impurity as the main root cause for improved cell performance and altered glycosylation profiles. This study sought to investigate the impact of iron raw material impurities within another medium platform, namely EX-CELL® Advanced CHO Fed-Batch-Medium. As opposed to previously published results, in this platform, copper instead of manganese impurity present within the used ferric ammonium citrate (FAC) iron source was responsible for an improved cell performance of a CHOZN® cell line and a slight difference in CQAs of the produced recombinant protein. The use of tightly controlled raw material specifications or the use of low impurity iron sources is therefore crucial to minimize the impact of impurities on cell performance in any CCM platform and thereby guarantee consistent and reproducible cell culture processes.

Capsicumicine, a New Bioinspired Peptide from Red Peppers Prevents Staphylococcal Biofilm In Vitro and In Vivo via a Matrix Anti-Assembly Mechanism of Action.

Staphylococci are pathogenic biofilm-forming bacteria and a source of multidrug resistance and/or tolerance causing a broad spectrum of infections. These bacteria are enclosed in a matrix that allows them to colonize medical devices, such as catheters and tissues, and that protects against antibiotics and immune systems. Advances in antibiofilm strategies for targeting this matrix are therefore extremely relevant. Here, we describe the development of the Capsicum pepper bioinspired peptide "capsicumicine." By using microbiological, microscopic, and nuclear magnetic resonance (NMR) approaches, we demonstrate that capsicumicine strongly prevents methicillin-resistant Staphylococcus epidermidis biofilm via an extracellular "matrix anti-assembly" mechanism of action. The results were confirmed in vivo in a translational preclinical model that mimics medical device-related infection. Since capsicumicine is not cytotoxic, it is a promising candidate for complementary treatment of infectious diseases. IMPORTANCE Pathogenic biofilms are a global health care concern, as they can cause extensive antibiotic resistance, morbidity, mortality, and thereby substantial economic loss. So far, no effective treatments targeting the bacteria in biofilms have been developed. Plants are constantly attacked by a wide range of pathogens and have protective factors, such as peptides, to defend themselves. These peptides are common components in Capsicum baccatum (red pepper). Here, we provide insights into an antibiofilm strategy based on the development of capsicumicine, a natural peptide that strongly controls biofilm formation by Staphylococcus epidermidis, the most prevalent pathogen in device-related infections.

Degradation Products of Tryptophan in Cell Culture Media: Contribution to Color and Toxicity.

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.

Adherent and suspension baby hamster kidney cells have a different cytoskeleton and surface receptor repertoire.

Animal cell culture, with single cells growing in suspension, ideally in a chemically defined environment, is a mainstay of biopharmaceutical production. The synthetic environment lacks exogenous growth factors and usually requires a time-consuming adaptation process to select cell clones that proliferate in suspension to high cell numbers. The molecular mechanisms that facilitate the adaptation and that take place inside the cell are largely unknown. Especially for cell lines that are used for virus antigen production such as baby hamster kidney (BHK) cells, the restriction of virus growth through the evolution of undesired cell characteristics is highly unwanted. The comparison between adherently growing BHK cells and suspension cells with different susceptibility to foot-and-mouth disease virus revealed differences in the expression of cellular receptors such as integrins and heparan sulfates, and in the organization of the actin cytoskeleton. Transcriptome analyses and growth kinetics demonstrated the diversity of BHK cell lines and confirmed the importance of well-characterized parental cell clones and mindful screening to make sure that essential cellular features do not get lost during adaptation.

S-Sulfocysteine - Investigation of cellular uptake in CHO cells.

For the generation of therapeutic proteins in cell culture, high producing clones are used. These clones have a high demand in amino acids to support cell growth and productivity. l-cysteine (Cys) is critical in highly concentrated feeds due to low stability of Cys and low solubility of the oxidation product cystine at neutral pH. S-sulfocysteine (SSC) was developed to substitute the Cys source and fed-batch experiments using SSC showed good cellular performance regarding viable cell density and titer, indicating uptake and metabolization of SSC by Chinese hamster ovary cells. However, the responsible transporter allowing cellular uptake remains unclear and was studied in this work. Due to the structure similarity of SSC with cystine and glutamate, it was proposed that the cystine/glutamate antiporter (xc-) allows cellular uptake of SSC. The uptake was assessed via transporter inhibition using sulfasalazine and transporter overexpression using either sulforaphane or sulforaphane-N-acetylcysteine during fed-batch experiments. Following daily addition of 50 µM and 100 µM sulfasalazine, the extracellular SSC concentration was increased by 65 % and 177 % respectively, suggesting a reduced uptake due to xc- inhibition. In contrast, enhanced transporter activity through 15 µM sulforaphane and sulforaphane-N-acetylcysteine treatment, induced a 60 % and 52 % reduced extracellular SSC concentration, respectively. These inverse uptake results strongly suggest that xc- is facilitating the transport of SSC.

Synthesis and antileukemic activity of an ursolic acid derivative: A potential co-drug in combination with imatinib.

Imatinib, a specific Bcr-Abl tyrosine kinase inhibitor, is the most commonly used drug in the treatment of chronic myeloid leukemia. However, optimal response is not achieved in up to 33% of patients. Therefore, development of novel therapeutic strategies for chronic myeloid leukemia is critical. Betulinic (1) and ursolic (2) acids are natural pentacyclic triterpenes that exhibit antileukemic activities. In this study, we evaluated the effects of pharmacomodulations at the C-3 position of the triterpene moiety of betulinic and ursolic acids on their activity against K562 leukemia cells. Six new derivatives (1a-2c) were synthesized and evaluated for pro-apoptotic and anti-proliferative effects in mammalian and leukemic cells. 2c derivative containing an amine group at the C-3 position of ursolic acid was the most active against leukemia cells with an IC50 value of 5.2 µM after 48 h of treatment. 2c did not exhibit cytotoxic effects against VERO and HepG2 cells and human lymphocytes, showing a good selectivity index for cancer over normal cells. Induced cell death by apoptosis via caspases 3 and 8, and also caused cell cycle arrest as evidenced by accumulation of cells in the G1 phase and decreased cell population in the G2 phase. Furthermore, co-treatment of 2c with imatinib, the chemotherapy drug most commonly used to treat leukemia, resulted in a synergistic effect. Our findings provide a strong rationale for further investigation of combination therapy using the 2c derivative and imatinib in pre-clinical studies.

Rapid size-exclusion high performance liquid chromatography method for the quality control of amyloid-ß oligomers.

Amyloid-ß (Aß) dysmetabolism is thought to be the main trigger for neurodegenerative events in Alzheimer's disease (AD). In particular, soluble Aß oligomers (AßOs) are proposed as key mediators of synaptic and cognitive dysfunction in AD. Over the past few decades, AßOs prepared from synthetic Aß have been widely applied in vitro and in vivo, the so-called chemical models of AD, uncovering their multiple neurotoxic mechanisms. However, the lack of a reliable quality control (QC) for synthetic AßOs may reflect poor experimental reproducibility. In keeping with this, we optimized and validated a rapid and reproducible SECHPLC method using fluorescence detection for the QC of synthetic AßOs. Our analytical method offers an unprecedent alternative to improve the reproducibility of AD chemical models.

Impact of iron raw materials and their impurities on CHO metabolism and recombinant protein product quality.

Cell culture medium (CCM) composition affects cell growth and critical quality attributes (CQAs) of monoclonal antibodies (mAbs) and recombinant proteins. One essential compound needed within the medium is iron because of its central role in many cellular processes. However, iron is also participating in Fenton chemistry leading to the formation of reactive oxygen species (ROS) causing cellular damage. Therefore, this study sought to investigate the impact of iron in CCM on Chinese hamster ovary (CHO) cell line performance, and CQAs of different recombinant proteins. Addition of either ferric ammonium citrate (FAC) or ferric citrate (FC) into CCM revealed major differences within cell line performance and glycosylation pattern, whereby ammonium was not involved in the observed differences. Inductively coupled plasma mass spectrometry (ICP-MS) analysis identified varying levels of impurities present within these iron sources, and manganese impurity rather than iron was proven to be the root cause for increased cell growth, titer, and prolonged viability, as well as altered glycosylation levels. Contrary effects on cell performance and protein glycosylation were observed for manganese and iron. The use of low impurity iron raw material is therefore crucial to control the effect of iron and manganese independently and to support and guarantee consistent and reproducible cell culture processes.

Lactoyl leucine and isoleucine are bioavailable alternatives for canonical amino acids in cell culture media.

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.

Use of 5-Thio-L-Fucose to modulate binding affinity of therapeutic proteins.

The reduction of antibody core-fucosylation is known to enhance antibody-dependent cellular cytotoxicity (ADCC). In this study, 5-Thio-l-Fucose (ThioFuc) was investigated as a media and feed supplement for modulating the fucosylation profile of therapeutic proteins and, thereby, improving the resulting effector functions. Glycan analysis of five different therapeutic proteins produced by a diverse set of Chinese hamster ovary cell lines demonstrated a clone dependent impact of ThioFuc treatment. Using rituximab as a model, an efficient dose- and time-dependent reduction of core-fucosylation up to a minimum of 5% were obtained by ThioFuc. Besides a concomitant increase in the afucosylation level up to 48%, data also revealed up to 47% incorporation of ThioFuc in place of core-fucosylation. In accordance with the glycan data, antibodies produced in the presence of ThioFuc revealed an enhanced FcγRIIIa binding up to 7.7-fold. Furthermore, modified antibodies subjected to a cell-based ADCC reporter bioassay proved to exert both a 1.5-fold enhanced ADCC efficacy and 2.6-fold enhancement in potency in comparison to their native counterparts-both of which contribute to an improvement in the ADCC activity. In conclusion, ThioFuc is a potent fucose derivative with potential applications in drug development processes.

Targeted Modification of the Foot-And-Mouth Disease Virus Genome for Quick Cell Culture Adaptation.

Foot-and-mouth disease virus (FMDV) causes the highly contagious foot-and-mouth disease, which is characterized by the appearance of vesicles in and around the mouth and feet of cloven-hoofed animals. BHK-21 cells are the cell line of choice for the propagation of FMDV for vaccine production worldwide but vary in their susceptibility for different FMDV strains. Previous studies showed that the FMDV resistance of a certain BHK cell line can be overcome by using a closely related but permissive cell line for the pre-adaptation of the virus, but the adapted strains were found to harbor several capsid mutations. In this study, these adaptive mutations were introduced into the original Asia-1 Shamir isolate individually or in combination to create a panel of 17 Asia-1 mutants by reverse genetics and examine the effects of the mutations on receptor usage, viral growth, immunogenicity and stability. A single amino acid exchange from glutamic acid to lysine at position 202 in VP1 turned out to be of major importance for productive infection of the suspension cell line BHK-2P. In consequence, two traditionally passage-derived strains and two recombinant viruses with a minimum set of mutations were tested in vivo. While the passaged-derived viruses showed a reduced particle stability, the genetically modified viruses were more stable but did not confer a protective immune response against the original virus isolate.

Reactivity and degradation products of tryptophan in solution and proteins.

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.

Modeling Diet-Induced Metabolic Syndrome in Rodents.

Standardized animal models represent one of the most valuable tools available to understand the mechanism underlying the metabolic syndrome (MetS) and to seek for new therapeutic strategies. However, there is considerable variability in the studies conducted with this essential purpose. This review presents an updated discussion of the most recent studies using diverse experimental conditions to induce MetS in rodents with unbalanced diets, discusses the key findings in metabolic outcomes, and critically evaluates what we have been learned from them and how to advance in the field. The study includes scientific reports sourced from the Web of Science and PubMed databases, published between January 2013 and June 2020, which used hypercaloric diets to induce metabolic disorders, and address the impact of the diet on metabolic parameters. The collected data are used as support to discuss variables such as sex, species, and age of the animals, the most favorable type of diet, and the ideal diet length to generate metabolic changes. The experimental characteristics propose herein improve the performance of a preclinical model that resembles the human MetS and will guide researchers to investigate new therapeutic alternatives with confidence and higher translational validity.

Quinolines derivatives as promising new antifungal candidates for the treatment of candidiasis and dermatophytosis.

Fungal infections have emerged as a current serious global public health problem. The main problem involving these infections is the expansion of multidrug resistance. Therefore, the prospection of new compounds with efficacy antifungal becomes necessary. Thus, this study evaluated the antifungal profile and toxicological parameters of quinolines derivatives against Candida spp. and dermatophyte strains. As a result, a selective anti-dermatophytic action was demonstrated by compound 5 (geometric means (GM = 19.14 µg ml-1)). However, compounds 2 (GM = 50 µg ml-1) and 3 (GM = 47.19 µg ml-1) have presented only anti-Candida action. Compounds 3 and 5 did not present cytotoxic action. Compound 5 did not produce dermal and mucosal toxicity. In addition, this compound showed the absence of genotoxic potential, suggesting safety for topical and systemic use. Quinolines demonstrated a potent anti-dermatophytic and anti-yeast action. Moreover, compound 5 presented an excellent toxicological profile, acting as a strong candidate for the development of a new effective and safe compound against dermatophytosis of difficult treatment.