Consequences of trace metal variability and supplementation on Chinese hamster ovary (CHO) cell culture performance: A review of key mechanisms and considerations

Trace metals are supplied to chemically-defined media (CDM) for optimal Chinese hamster ovary (CHO) cell culture performance during the production of monoclonal antibodies and other therapeutic proteins. However, lot-to-lot and vendor-to-vendor variability in raw materials consequently leads to an imbalance of trace metals that are supplied to CDM. This imbalance can yield detrimental effects rooted in several primary mechanisms and pathways including oxidative stress, apoptosis, lactate accumulation, and unfavorable glycan synthesis. Recent research endeavors involve supplying zinc, copper, and manganese to CDM in excess to further maximize culture productivity and product quality. These treatments significantly impact critical quality attributes and furthermore highlight the degree to which trace metal availability can affect CHO cell culture performance. This review highlights the role of trace metal variability, supplementation, and interplay on key cellular mechanisms responsible for overall culture performance and the production and quality of therapeutic proteins.     

Trace element uptake by L-cells as a function of trace elements in a synthetic growth medium

The concentration of trace elements in L-cells has been studied as a function of the trace metal content of the growth medium. Cells were cultured in synthetic media which contained varying trace amounts of the elements manganese, iron, cobalt, copper, zinc and molybdenum. The cellular concentration of the elements potassium, iron, copper and zinc were then determined. It was found that the cell accumulates trace metals at a different rate than they are made available. Deficiencies in zinc could be “induced” in the cell by increasing the concentration of iron, manganese and cobalt; cellular iron deficiencies were observed at larger medium concentrations of zinc, manganese, copper and cobalt. Trace metal uptake by the cell was seen to parallel the utilization by multicellular organisms.     

Micronutrient Requirements for Growth and Hydrocarbon Production in the Oil Producing Green Alga Botryococcus braunii (Chlorophyta)

The requirements of micronutrients for biomass and hydrocarbon production in Botryococcus braunii UTEX 572 were studied using response surface methodology. The concentrations of four micronutrients (iron, manganese, molybdenum, and nickel) were manipulated to achieve the best performance of B. braunii in laboratory conditions. The responses of algal biomass and hydrocarbon to the concentration variations of the four micronutrients were estimated by a second order quadratic regression model. Genetic algorithm calculations showed that the optimal level of micronutrients for algal biomass were 0.266 μM iron, 0.707 μM manganese, 0.624 μM molybdenum and 3.38 μM nickel. The maximum hydrocarbon content could be achieved when the culture media contained 10.43 μM iron, 6.53 μM manganese, 0.012 μM molybdenum and 1.73 μM nickel. The validation through an independent test in a photobioreactor suggests that the modified media with optimised concentrations of trace elements can increase algal biomass by 34.5% and hydrocarbon by 27.4%. This study indicates that micronutrients play significant roles in regulating algal growth and hydrocarbon production, and the response surface methodology can be used to optimise the composition of culture medium in algal culture.     

Impact of dynamic online fed-batch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures

As we pursue the means to improve yields to meet growing therapy demands, it is important to examine the impact of process control on glycosylation patterns to ensure product efficacy and consistency. In this study, we describe a dynamic on-line fed-batch strategy based on low glutamine/glucose concentrations and its impact on cellular metabolism and, more importantly, the productivity and N-glycosylation quality of a model recombinant glycoprotein, interferon gamma (IFN-gamma). We found that low glutamine fed-batch strategy enabled up to 10-fold improvement in IFN-gamma yields, which can be attributed to reduced specific productivity of ammonia and lactate. Furthermore, the low glutamine concentration (0.3 mM) used in this fed-batch strategy could maintain both the N-glycosylation macro- and microheterogeneity of IFN-gamma. However, very low glutamine (<0.1 mM) or glucose (<0.70 mM) concentrations can lead to decreased sialylation and increased presence of minor glycan species consisting of hybrid and high-mannose types. This shows that glycan chain extension and sialylation can be affected by nutrient limitation. In addition to nutrient limitation, we also found that N-glycosylation quality can be detrimentally affected by low culture viability. IFN-gamma purified at low culture viability had both lower sialylation as well as glycans of lower molecular masses, which can be attributed to extensive degradation by intracellular glycosidases released by cytolysis. Therefore, in order to maintain good N-glycosylation quality, there is a need to consider both culture viability and nutrient control setpoint in a nutrient-limiting fed-batch culture strategy. A greater understanding of these major factors that affect N-glycosylation quality would surely facilitate future development of effective process controls.     

Probing of C-terminal lysine variation in a recombinant monoclonal antibody production using Chinese hamster ovary cells with chemically defined media

C-terminal lysine (C-K) variants are commonly observed in therapeutic monoclonal antibodies and recombinant proteins. Heterogeneity of C-K residues is believed to result from varying degree of proteolysis by endogenous carboxypeptidase(s) during cell culture production. The achievement of batch-to-batch culture performance and product quality reproducibility is a key cell culture development criterion. Understanding the operational parameters affecting C-K levels provides valuable insight into the cell culture process. A CHO cell line X expressing a recombinant antibody was selected as the model cell line due to the exhibited sensitivity of its C-K level to the process conditions. A weak cation exchange chromatography (WCX) method with or without carboxypeptidase B (CpB) treatment was developed to monitor the C-K level for in-process samples. The effects of operating conditions (i.e., temperature and culture duration) and media trace elements (copper and zinc) on C-K variants were studied. The dominant effect on C-K level was identified as the trace elements concentration. Specifically, increased C-K levels were observed with increase of copper concentration and decrease of zinc concentration in chemically defined medium. Further, a hypothesis for C-K processing with intracellular and extracellular carboxypeptidase activity was proposed, based on preliminary intracellular carboxypeptidase Western blot results and the extracellular HCCF holding study.     

Modulation of mAb quality attributes using microliter scale fed‐batch cultures

A high‐throughput DoE approach performed in a 96‐deepwell plate system was used to explore the impact of media and feed components on main quality attributes of a monoclonal antibody. Six CHO‐S derived clonal cell lines expressing the same monoclonal antibody were tested in two different cell culture media with six components added at three different levels. The resulting 384 culture conditions including controls were simultaneously tested in fed‐batch conditions, and process performance such as viable cell density, viability, and product titer were monitored. At the end of the culture, supernatants from each condition were purified and the product was analyzed for N‐glycan profiles, charge variant distribution, aggregates, and low molecular weight forms. The screening described here provided highly valuable insights into the factors and combination of factors that can be used to modulate the quality attributes of a molecule. The approach also revealed specific intrinsic differences of the selected clonal cell lines ‐ some cell lines were very responsive in terms of changes in performance or quality attributes, whereas others were less affected by the factors tested in this study. Moreover, it indicated to what extent the attributes can be impacted within the selected experimental design space. The outcome correlated well with confirmations performed in larger cell culture volumes such as small‐scale bioreactors. Being fast and resource effective, this integrated high‐throughput approach can provide information which is particularly useful during early stage cell culture development. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:571–583, 2014     

Effect of the cultivation conditions on alkaloid biosynthesis by Penicillium gorlenkoanum

The effect of a carbohydrate component of the medium, trace elements and aeration on biosynthesis of the alkaloids costaclavine and epicostaclavine was studied with Penicillium gorlenkoanum. Alkaloid biosynthesis was shown to depend on the nature of a carbohydrate component: virtually no alkaloids were accumulated in media with glucose and fructose although these were synthesized at a high rate in a medium with mannitol. The quantity of synthesized alkaloids and the dynamics of the biosynthesis depended on carbohydrate concentration. The growth and alkaloid synthesis were influenced by traces of zinc, iron, copper and manganese. A more intensive aeration stimulated biomass accumulation but suppressed alkaloid biosynthesis.     

Trace elements status in multinodular goiter

Importance of iodine and selenium in thyroid metabolism is well known, but the roles of other essential trace elements including copper, zinc, manganese and iron on thyroid hormone homeostasis remain unclear. The aim of this study was to investigate the status of those trace elements in benign thyroid diseases and evaluate possible links between trace element concentrations and thyroid hormones.The study group was composed of 25 patients with multinodular goiter. Concentrations of thyroid hormones (plasma-free thyroxine, FT₄; free triiodothyronine, FT₃; and thyrotropin, TSH), selenium, copper, zinc, manganese and iron in plasma, and urinary iodine were determined. The results were compared with those of a healthy control group (n=20) with no thyroid disorder.A mild iodine deficiency was observed in the patients with multinodular goiter whereas urinary iodine levels were in the range of “normal” values in healthy controls. All patients were euthyroid, and their thyroid hormone concentrations were not significantly different from the control group. Plasma selenium, zinc and iron concentrations did not differ from controls, while copper and manganese levels were found to be significantly higher in the patients with multinodular goiter indicating links between these trace elements and thyroid function and possibly in development of goiter. Besides iodine, there was a significant correlation between plasma copper concentration and FT₃/FT₄ ratio.     

Trace element budget in an African savannah ecosystem

The concentration of selected trace elements (Co, Cu, Fe, Mn, Mo, Se, and Zn) were analysed in soils, grass, bush, and tree samples from the Mole National Park, Ghana. The distribution of the essential nutrients: cobalt, copper, manganese, and selenium is controlled by bedrock geology, whereas iron, molybdenum, and zinc distribution is controlled by soil and hydrological processes. In the soils, iron, manganese, and cobalt are largely fixed in the mineral fraction while most of the copper, molybdenum, and selenium in the soils can be extracted by disodium ethylenediaminetetracetate. Copper, cobalt, and manganese appear to be preferentially concentrated in grass species while molybdenum and selenium are concentrated in browse plants. Variations in uptake exist between wet and dry seasons with all trace elements studied, except iron and manganese, showing a marked increased availability in the wet season and increased concentration in the residual fraction of the mineral and organic soils in the dry season. In the dry season the plant concentration of molybdenum and selenium decreased while copper and zine showed increased concentrations and this may be related to a lower pH of the groundwaters at this time. A budget of metal input and output in the ecosystem at Mole has been computed. From this potential dietary deficiencies in cobalt can be observed, however for other metals soil and plant concentrations are sufficient to prevent straightforward deficiencies while the concentrations of molybdenum and selenium are sufficiently low to be considered safe.     

Framework for the rapid optimization of soluble protein expression in Escherichia coli combining microscale experiments and statistical experimental design

A major bottleneck in drug discovery is the production of soluble human recombinant protein in sufficient quantities for analysis. This problem is compounded by the complex relationship between protein yield and the large number of variables which affect it. Here, we describe a generic framework for the rapid identification and optimization of factors affecting soluble protein yield in microwell plate fermentations as a prelude to the predictive and reliable scaleup of optimized culture conditions. Recombinant expression of firefly luciferase in Escherichia coli was used as a model system. Two rounds of statistical design of experiments (DoE) were employed to first screen (D-optimal design) and then optimize (central composite face design) the yield of soluble protein. Biological variables from the initial screening experiments included medium type and growth and induction conditions. To provide insight into the impact of the engineering environment on cell growth and expression, plate geometry, shaking speed, and liquid fill volume were included as factors since these strongly influence oxygen transfer into the wells. Compared to standard reference conditions, both the screening and optimization designs gave up to 3-fold increases in the soluble protein yield, i.e., a 9-fold increase overall. In general the highest protein yields were obtained when cells were induced at a relatively low biomass concentration and then allowed to grow slowly up to a high final biomass concentration, >8 g.L-1. Consideration and analysis of the model results showed 6 of the original 10 variables to be important at the screening stage and 3 after optimization. The latter included the microwell plate shaking speeds pre- and postinduction, indicating the importance of oxygen transfer into the microwells and identifying this as a critical parameter for subsequent scale translation studies. The optimization process, also known as response surface methodology (RSM), predicted there to be a distinct optimum set of conditions for protein expression which could be verified experimentally. This work provides a generic approach to protein expression optimization in which both biological and engineering variables are investigated from the initial screening stage. The application of DoE reduces the total number of experiments needed to be performed, while experimentation at the microwell scale increases experimental throughput and reduces cost.     

Characterization of soy protein hydrolysates and influence of its iron content on monoclonal antibody production by a murine hybridoma cell line

A challenging aspect with the use of protein hydrolysates in commercial manufacturing processes of recombinant therapeutic proteins is their impacts on the protein production due to a lack of understanding of batch-to-batch variability. Soy hydrolysates variability and its impact on fed-batch production of a recombinant monoclonal antibody (mAb) expressed in Sp2/0 cells were studied using 37 batches from the same vendor. The batch-to-batch variability of soy hydrolysates impacted cell growth, titer and product quality. Physicochemical characterization of batches confirmed that soy hydrolysates are mainly a source of amino acids and peptides containing lower amounts of other components such as carbohydrates and chemical elements in cell culture media. Soy hydrolysates composition of different batches was consistent except for trace elements. Statistical analyses identified iron as a potential marker of a poor process performance. To verify this correlation, two forms of iron, ferric ammonium citrate and ferrous sulfate, were added to a batch of soy hydrolysates associated to a low level of iron during cell culture. Both forms of iron reduced significantly cell growth, mAb titer and increased level of the acidic charge variants of the mAb. Consequently, trace element composition of soy hydrolysates or of all incoming raw materials might lead to significant impacts on process performance and product quality and therefore need to be tightly controlled.     

Manganese, copper, zinc, and iron concentrations and subcellular distribution in two types of skeletal muscle

To clarify trace element distribution in red and white muscle, and to verify two populations of muscle mitochondria, the iron, zinc, copper, and manganese concentrations of whole muscle and their subcellular fractions were determined. The iron, zinc, copper, and manganese concentrations of red muscle were 1.83, 4.31, 2.05, and 1.67 times higher than those of white muscle, respectively. In skeletal muscle subcellular distribution or iron, zinc, and copper were entirely different and that of manganese was relatively similar as compared with those in liver reported previously. The pattern of mineral distribution in all fractions of red muscle was similar to that of white muscle, but their concentrations in some fractions were different between red and white muscle, e.g., iron, zinc, and manganese in supernatant fraction and copper in nuclear and microsomal fractions. The difference between subsarcolemmal and interfibrillar mitochondria were ascertained by the distribution of trace elements.     

Determination of Trace Elements in Jinqi,a Traditional Chinese Medicine

We have determined the trace element composition of Jingi, a common remedy used in traditional Chinese medicine, using atomic absorption spectrophotometer (AAS). The concentration of the trace elements analyzed in this medicine decreases in the order: zinc > manganese > chromium > magnesium > copper > iron > lead > nickel > vanadium. We suggest that these trace elements may play a direct or indirect role in the hypoglycemic properties of Jinqi. The three plants used as main ingredients in the preparation of this recipe should be planted in a lead-free soil rich in zinc, manganese, chromium, magnesium, and vanadium.     

Changes in trace element concentrations in hair of growing children

Head hair concentrations of zinc, copper, manganese, and iron from a total of 418 subjects (154 male and 264 female) aged between 6 mo and 20 yr were measured mainly with flameless atomic absorption spectrophotometry. Only zinc analysis of a part of the female samples (n=140) were analyzed with inductively coupled plasma-atomic emission spectrometry. The two analytical methods showed close agreement. The mean concentration of copper and manganese were significantly higher in male subjects than in female subjects. The trace element concentrations in hair varied with the subject’s age. Zinc concentration in hair decreased from 6 mo to 14 yr in the male subjects and decreased from 6 mo to 12 yr in the female subjects. Then, the concentrations increased gradually to 20 yr in the both sexes. Age-dependent variations of copper and manganese concentrations in hair showed similar trends to those of zinc. The results of this study suggest that a higher concentration in the diet of these trace elements may be required for growing children, especially in the period of adolescence.     

Measurement of zinc, copper, manganese, and iron concentrations in hair of pituitary dwarfism patients using flameless atomic absorption spectrophotometry

Pituitary dwarfism (hGHD) is known to be associated with trace element deficiency, which causes improper functioning of the involved endocrine system. Previously, we reported on the head hair concentrations of zinc, copper, manganese, and iron from a total of 418 normal subjects (154 male and 264 female). In this report, we analyzed the head hair concentrations of the same four trace metals of 103 hGHD children (60 male and 43 female) under treatment with human growth hormone (hGH). These subjects ranged in age from 5 to 18 yr. The results were compared with 338 agematched normal subjects (120 male and 218 female). Both male and female hGHD showed approx 1.7 times higher zinc concentrations than normal subjects. Cheruvanky et al. reported a similar trend but with a slightly lower difference between hGHD and normal subjects. The average copper content in the hair of both male and female subjects also showed higher values for the hGHD than for the normal subjects, a trend similar to the values reported by Teraoka et al. In the case of manganese, the concentrations in hair of the hGHD were approx 50% of the values in the normal subjects. Head hair concentrations of iron in the hGHD were commensurate with the normal subjects. Because the content of trace elements in hair varies with the age of subjects, as a control, we investigated the head hair concentration of zinc from 20 healthy girls ranging in age from 10 to 18 yr. The average zinc concentration decreased from 10 to 12 yr, but no clear relation to age was observed from 13 yr and older. These trends were similar to our previous report. The zinc concentration in hair and body weight gain over a year was negatively correlated. The age variation in the content of zinc, copper, manganese, and iron in hair was measured comparing hGHD with the normal subjects in various ages. Concerning the zinc-level variation of hGHD and normal subjects, there were conspicuous differences between hGHD and normal subjects. For copper, the variations in concentration with age were similar to zinc. Regarding the age variations for manganese, hGHD had lower concentrations in hair compared to the normal subjects throughout adolescence (11–18 yr). We have studied the effects between the hair and these trace element concentrations in hGHD before and after hGH administration. These results suggest that hGH affects the metabolism of these trace elements.     

Effect of phosphorus and zinc on the growth and phosphorus,zinc, copper,iron and manganese nutrition of rice

Summary A greenhouse experiment was conducted to study the effect of phosphorus and zinc application, in three lowland alluvial rice soils (Haplustalf) on the growth of rice and the concentration of phosphorus, zinc, copper, iron and manganese in shoots and roots. The results showed that application of phosphorus and zinc significantly increased the dry matter yield of shoots, grains and roots. Application of phosphorus caused a decrease in the concentration of zinc, copper, iron and manganese both in shoots and roots. Application of zinc also similarly lowered the concentration of phosphorus, copper and iron, but increased that of manganese in shoots and roots. The decrease in the concentration of the elements in the shoots was not due to dilution effect or to the reduced rate of translocation of the elements from the roots to tops. This has been attributed more to the changes in the availability of the elements in soil resulting from the application of phosphorus and zinc.     

Metals in Wine—Impact on Wine Quality and Health Outcomes

Metals in wine can originate from both natural and anthropogenic sources, and its concentration can be a significant parameter affecting consumption and conservation of wine. Since metallic ions have important role in oxide-reductive reactions resulting in wine browning, turbidity, cloudiness, and astringency, wine quality depends greatly on its metal composition. Moreover, metals in wine may affect human health. Consumption of wine may contribute to the daily dietary intake of essential metals (i.e., copper, iron, and zinc) but can also have potentially toxic effects if metal concentrations are not kept under allowable limits. Therefore, a strict analytical control of metal concentration is required during the whole process of wine production. This article presents a critical review of the existing literature regarding the measured metal concentration in wine, methods applied for their determination, and possible sources, as well as their impact on wine quality and human health. The main focus is set on aluminum, arsenic, cadmium, chromium, copper, iron, manganese, nickel, lead, and zinc, as these elements most often affect wine quality and human health.     

Are Additional Trace Elements Necessary in Total Parenteral Nutrition for Patients with Esophageal Cancer Receiving Cisplatin-Based Chemotherapy?

It is known that cisplatin induces the excretion of zinc from the urine and thereby reduces its serum concentration. However, the fluctuation of these trace elements during or after cisplatin-based chemotherapy has not been evaluated. To answer this question, we performed a clinical study in esophageal cancer patients undergoing cisplatin-based chemotherapy. Eighteen patients with esophageal cancer who were not able to swallow food or water orally due to complete stenosis of the esophagus were evaluated. The patients were divided into a control group [total parenteral nutrition (TPN) alone for 28?days, ten cases] and an intervention group (TPN with additional trace elements for 28?days, eight cases). The serum concentrations of zinc, iron, copper, manganese, triiodothyronin (T3), and thyroxin (T4), as alternative indicators of iodine, were measured on days?0, 14, and 28 of treatment, and statistically analyzed on day?28. In the control group, the serum concentration of copper was significantly decreased from 135.4 (day?0) to 122.1???g/ml (day?14), and finally to 110.6???g/ml (day?28, p?=?0.015). The concentration of manganese was also significantly decreased from 1.34 (day?0) to 1.17???g/ml (day?14) and finally to 1.20 (day?28, p?=?0.049). The levels of zinc, iron, T3, and T4 were not significantly changed. In the intervention group, the supplementation with trace elements successfully prevented these decreases in their concentrations. TPN with supplementary trace elements is preferable and recommended for patients who are undergoing chemotherapy in order to maintain the patients?? nutrient homeostasis.     

Assessment of trace elements in human brain using inductively coupled plasma mass spectrometry

Recent brain research reveals a major role of trace elements in various diseases such as multiple sclerosis, Alzheimer's and Wilson's disease. The majority of published tissue concentrations dates back decades, and was assessed with various methods. Little is known about hemispherical differences, the correlation of trace elements or age-dependent changes in the human brain. Thus, the aim of this study was to examine trace element concentrations in different human brain regions after whole brain formalin fixation.549 samples of 13 brain regions were investigated in 11 deceased subjects without known history of brain pathology. Regional wet-to-dry mass ratios and concentrations of iron, copper, magnesium, manganese, calcium and zinc were determined using inductively coupled plasma mass spectrometry.Cortical gray matter revealed higher water content (wet-to-dry mass ratios 5.84–6.40) than white matter regions (wet-to-dry mass ratios 2.95–3.05). Element concentrations displayed specific regional differences. Good linear correlation of concentrations between elements was found for iron/copper as well as for manganese/magnesium (Spearman's rank correlation coefficient 0.74 and 0.65, respectively). Significant inter-hemispherical differences were found for copper in occipital white matter, for magnesium and calcium in putamen and for iron and copper in temporal white matter. An age dependent increase was seen in cortical gray matter for calcium, for magnesium in all regions except in cortical gray matter, for copper in substantia nigra and for zinc in occipital cortex.The presented trace element concentrations can serve as a fundamental basis for further brain research. Wet-to-dry mass ratios allow a comparison with reference data from other studies.