Validation of Rapi-Fluor method for glycan profiling and application to commercial antibody drugs.

N-glycans influence the activity of antibody drugs such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Thus, glycan profiling is considered a critical quality attribute (CQA) and requires routine and comprehensive monitoring. In this report, we validate the new glycan profiling method called Rapi-Fluor method, which reduced the sample preparation time and increased the FLR and MS intensities compared with conventional 2-AB method. Optimized glycan release, labeling, hydrophilic interaction liquid chromatography (HILIC) enrichment, and HILIC separation resulted in low variation and short preparation time. The method evaluated for human IgG standard varied from 100 µg/mL to 4000 µg/mL in 25 µL of water. The determination of coefficient (r2 > 0.9992), recovery (88.992% ~ 111.198%), limit of detection (LOD < 193.274 µg/mL), limit of quantification (LOQ < 585.679 µg/mL), and precision (Intra-day < 2.317%RSD and Inter-day < 4.287%RSD) were evaluated with four major glycans from antibody drugs. In addition, the method was used for glycan profiling of five different commercial antibodies. The method yielded precise results for IgG glycan analysis and demonstrated effective glycan profiling of commercial antibody drugs.

Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells.

Chinese hamster ovary (CHO) cells are the most common cell line used for the production of therapeutic proteins including monoclonal antibodies (mAbs). Host cell proteins (HCPs), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant CHO (rCHO) cells, potentially impairing product quality. In an effort to maintain good mAb quality during the cultures, HCPs accumulated extracellularly in batch and fed-batch cultures of a mAb-producing rCHO cell line were identified and quantified by nanoflow liquid chromatography-tandem mass spectrometry, followed by their gene ontology and functional analysis. Due to higher cell concentration and longer culture duration, more HCPs were identified and quantitated in fed-batch culture (2145 proteins identified and 1673 proteins quantified) than in batch culture (1934 proteins identified and 1486 proteins quantified). Clustering analysis of HCPs showed that the concentration profiles of HCPs affecting mAb quality (Lgmn, Ctsd, Gbl1, and B4galt1) correlated with changes in mAb quality attributes such as aggregation, charge variants, and N-glycosylation during the cultures. Taken together, the dataset of HCPs obtained in this study provides insights into determining the appropriate target proteins to be removed during both the cultures and purification steps for ensuring good mAb quality.

Reduction of ammonia and lactate through the coupling of glutamine synthetase selection and downregulation of lactate dehydrogenase-A in CHO cells.

Chinese hamster ovary (CHO) cell cultivation for production of therapeutic proteins is accompanied by production of metabolic wastes, mostly ammonia and lactate. To reduce ammonia production, the glutamine synthetase (GS) system was used to develop therapeutic monoclonal antibody (mAb)-producing CHO cells (SM-0.025). Additionally, the lactate dehydrogenase-A (LDH-A) was downregulated with shRNA to reduce lactate production in SM-0.025. The resulting mAb-producing cell lines (#2, #46, and #52) produced less ammonia than the host cell line during the exponential phase due to GS protein overexpression. LDH-A downregulation in SM-0.025 not only reduced lactate production but also further reduced ammonia production. Among the three LDH-A-downregulated clones, clone #2 had the highest mAb production along with significantly reduced specific lactate and ammonia production rates compared to those in SM-0.025. Waste reduction increased the galactosylation level of N-glycosylation, which improved mAb quality. LDH-A downregulation was also successfully applied to the host cell lines (CHO K1 and GS knockout CHO-K1). However, LDH-A downregulated host cells could not survive the pool-selection process wherein glutamine was excluded and methionine sulfoximine was added to the media. Taken together, LDH-A downregulation in the mAb-producing cell line generated with the GS system successfully reduced both ammonia and lactate levels, improving mAb galactosylation. However, LDH-A downregulation could not be applied to host cell lines because it hampered the selection process of the GS system.

Impaired functions of neural stem cells by abnormal nitric oxide-mediated signaling in an in vitro model of Niemann-Pick type C disease.

Nitric oxide (NO) has been implicated in the promotion of neurodegeneration. However, little is known about the relationship between NO and the self-renewal or differentiation capacity of neural stem cells (NSCs) in neurodegenerative disease. In this study, we investigated the effect of NO on self-renewal of NSCs in an animal model for Niemann-Pick type C (NPC) disease. We found that NO production was significantly increased in NSCs from NPC1-deficient mice (NPC1-/-), which showed reduced NSC self-renewal. The number of nestin-positive cells and the size of neurospheres were both significantly decreased. The expression of NO synthase (NOS) was increased in neurospheres derived from the brain of NPC1-/- mice in comparison to wild-type neurospheres. NO-mediated activation of glycogen synthase kinase-3beta (GSK3beta) and caspase-3 was also observed in NSCs from NPC1-/- mice. The self-renewal ability of NSCs from NPC1-/- mice was restored by an NOS inhibitor, L-NAME, which resulted in the inhibition of GSK3beta and caspase-3. In addition, the differentiation ability of NSCs was partially restored and the number of Fluoro-Jade C-positive degenerating neurons was reduced. These data suggest that overproduction of NO in NPC disease impaired the self-renewal of NSCs. Control of NO production may be key for the treatment of NPC disease.