Biologically Consistent Annotation of Metabolomics Data.

Annotation of metabolites remains a major challenge in liquid chromatography-mass spectrometry (LC-MS) based untargeted metabolomics. The current gold standard for metabolite identification is to match the detected feature with an authentic standard analyzed on the same equipment and using the same method as the experimental samples. However, there are substantial practical challenges in applying this approach to large data sets. One widely used annotation approach is to search spectral libraries in reference databases for matching metabolites; however, this approach is limited by the incomplete coverage of these libraries. An alternative computational approach is to match the detected features to candidate chemical structures based on their mass and predicted fragmentation pattern. Unfortunately, both of these approaches can match multiple identities with a single feature. Another issue is that annotations from different tools often disagree. This paper presents a novel LC-MS data annotation method, termed Biologically Consistent Annotation (BioCAn), that combines the results from database searches and in silico fragmentation analyses and places these results into a relevant biological context for the sample as captured by a metabolic model. We demonstrate the utility of this approach through an analysis of CHO cell samples. The performance of BioCAn is evaluated against several currently available annotation tools, and the accuracy of BioCAn annotations is verified using high-purity analytical standards.

Controlling trisulfide modification in recombinant monoclonal antibody produced in fed-batch cell culture.

Molecular heterogeneity was detected in a recombinant monoclonal antibody (IgG1 mAb) due to the presence of a trisulfide linkage generated by the post-translational insertion of a sulfur atom into disulfide bonds at the heavy-heavy and heavy-light junctions. This molecular heterogeneity had no observable effect on antibody function. Nevertheless, to minimize the heterogeneity of the IgG1 mAb from run-to-run, an understanding of the impact of cell culture process conditions on trisulfide versus disulfide linkage formation was desirable. To investigate variables that might impact trisulfide formation, cell culture parameters were varied in bench-scale bioreactor studies. Trisulfide analysis of the samples from these runs revealed that the trisulfide content in the bond between heavy and light chains varied considerably from <1% to 39%. Optimizing the culture duration and feeding strategy resulted in more consistent trisulfide levels. Cysteine concentration in the feed medium had a direct correlation with the trisulfide level in the product. Systematic studies revealed that cysteine in the feed and the bioreactor media was contributing hydrogen sulfide which reacted with the IgG1 mAb in the supernatant leading to the insertion of sulfur atom and formation of a trisulfide bond. Cysteine feed strategies were developed to control the trisulfide modification in the recombinant monoclonal antibody.

Using Metabolomics to Identify Cell Line-Independent Indicators of Growth Inhibition for Chinese Hamster Ovary Cell-based Bioprocesses.

Chinese hamster ovary (CHO) cells are widely used for the production of biopharmaceuticals. Efforts to improve productivity through medium design and feeding strategy optimization have focused on preventing the depletion of essential nutrients and managing the accumulation of lactate and ammonia. In addition to ammonia and lactate, many other metabolites accumulate in CHO cell cultures, although their effects remain largely unknown. Elucidating these effects has the potential to further improve the productivity of CHO cell-based bioprocesses. This study used untargeted metabolomics to identify metabolites that accumulate in fed-batch cultures of monoclonal antibody (mAb) producing CHO cells. The metabolomics experiments profiled six cell lines that are derived from two different hosts, produce different mAbs, and exhibit different growth profiles. Comparing the cell lines' metabolite profiles at different growth stages, we found a strong negative correlation between peak viable cell density (VCD) and a tryptophan metabolite, putatively identified as 5-hydroxyindoleacetaldehyde (5-HIAAld). Amino acid supplementation experiments showed strong growth inhibition of all cell lines by excess tryptophan, which correlated with the accumulation of 5-HIAAld in the culture medium. Prospectively, the approach presented in this study could be used to identify cell line- and host-independent metabolite markers for clone selection and bioprocess development.

Tryptophan oxidation catabolite, N-formylkynurenine, in photo degraded cell culture medium results in reduced cell culture performance.

Chemically defined media have been widely used in the biopharmaceutical industry to enhance cell culture productivities and ensure process robustness. These media, which are quite complex, often contain a mixture of many components such as vitamins, amino acids, metals and other chemicals. Some of these components are known to be sensitive to various stress factors including photodegradation. Previous work has shown that small changes in impurity concentrations induced by these potential stresses can have a large impact on the cell culture process including growth and product quality attributes. Furthermore, it has been shown to be difficult to detect these modifications analytically due to the complexity of the cell culture media and the trace level of the degradant products. Here, we describe work performed to identify the specific chemical(s) in photodegraded medium that affect cell culture performance. First, we developed a model system capable of detecting changes in cell culture performance. Second, we used these data and applied an LC-MS analytical technique to characterize the cell culture media and identify degradant products which affect cell culture performance. Riboflavin limitation and N-formylkynurenine (NFK), a tryptophan oxidation catabolite, were identified as chemicals which results in a reduction in cell culture performance.

Investigation of metabolic variability observed in extended fed batch cell culture.

A 13-day fed-batch IgG1 production process was developed by applying our proprietary chemically defined platform process. The process was highly reproducible with respect to cell growth and titer, but the cultures exhibited metabolic variability after 12 days of cultivation. This metabolic variability consisted of a subset of cultures exhibiting increased cell-specific glucose uptake rates and high lactate production rates (LPR) despite identical operating conditions. We investigated the causes of the metabolic variability by manipulating the rate at which feed medium was delivered. Overfeeding directly led to increased LPR. High LPR was found to be associated with increased mitochondrial membrane potential in a subset of cells, as measured through fluorescent staining, and feeding TCA cycle intermediates was found to prevent the high LPR phenotype. This supports the hypothesis that mitochondrial pathways are involved in inducing metabolic variability.