Evaluation of exogenous siRNA addition as a metabolic engineering tool for modifying biopharmaceuticals

Tummala, Seshu B.; Titus, Michael; Wilson, Lionel; Wang, Chunhua; Ciatto, Carlo; Thill, Greg; Foster, Donald J.; Li, Chen; Szabó, Zoltán; Guttman, András; Bettencourt, Brian R.; Jayaraman, Muthuswamy; Deroot, Jack; Kocisko, David A.; Pollard, Stuart; Charissé, Klaus; Kuchimanchi, Satya; Hinkle, Greg; Milstein, Stuart; Meyers, Rachel; Wu, Shiaw Lin; Karger, Barry L.; Rossomando, Anthony

doi:10.1002/btpr.1667

Cited by 8 publications

(9 citation statements)

References 34 publications

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“…Five milliliters of each sample was then centrifuged at 1,000 rpm to pellet the cells, with the pellet stored at −80 °C until needed. Prior to conducting this study, CHO‐DG44 cells were stably transfected with a previously reported plasmid vector containing an anti‐CD20 mAb gene sequence (bioreactor studies) or an empty plasmid vector without no transgene expressed (proteomic studies) containing the DHFR gene controlled by a cytomegalovirus promoter. Viable cell densities (VCDs) were measured using a Multisizer Coulter Counter (Beckman Coulter, Fullerton, CA).…”

Section: Methodsmentioning

confidence: 99%

A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells

Liu

Dai

Bones

et al. 2015

Biotechnology Progress

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A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO-DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO-DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA-based CHO-DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self-organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by upregulating NCK1 and down-regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial-and endoplasmic reticulum-mediated cell death pathways by up-regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production.

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Section: Methodsmentioning

confidence: 99%

A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells

Liu

Dai

Bones

et al. 2015

Biotechnology Progress

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“…RNAi-mediated mAb was manufactured at Alnylam Pharmaceuticals and provided as 1 mg/mL × 0.8 mL as previously described. 12 Rituximab was purchased by Alnylam from Imperial College, London and provided as 10 mg/mL × 0.5 mL. The samples were aliquoted as 10 μL per vial for rituximab (100 μg) and 100 μL per vial for RNAi-mediated mAb (100 μg) and stored at −80°C before analysis.…”

Section: Methodsmentioning

confidence: 99%

“…The reduction of the core fucose induced a 17-fold improvement in FcγRIIIa binding, and an increase in specific cell lysis by up to 30%, as determined in an ADCC assay. 12 Other modifications. MAbs with "Q" or "E" N-terminal amino acids could easily convert to pyro-Glu.…”

Section: Fc Glycosylationmentioning

confidence: 99%

“…[7][8][9][10][11] Thus, RNAi-mediated fucosyltransferase (FUT8) and GDP-man-4,6-dehydratase (GMDS) was used to produce anti-CD20 mAb for this purpose. 12 Although the aim was for a biobetter product, the overall structure, except for the level of the core fucose, was intended to be as similar as possible to the reference product to maintain the drug integrity.…”

Section: Introductionmentioning

confidence: 99%

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Comparability analysis of anti-CD20 commercial (rituximab) and RNAi-mediated fucosylated antibodies by two LC-MS approaches

Rossomando

et al. 2013

mAbs

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In developing biosimilar or biobetter products, comparability to the reference product is required to claim similar integrity or intended purpose. In this work, an anti-CD20 monoclonal antibody developed using RNA interference to decrease core fucosylation (RNAi-mediated) was comprehensively characterized by LC-MS and compared with the commercially-available anti-CD20 rituximab (MabThera (®) ). As anticipated, < 30% core fucose was found within the RNAi-produced molecule (compared with > 90% in rituximab), and the reduction in fucose resulting in a significant improvement in FcγRΙΙΙa binding and antibody-dependent cell-mediated cytotoxicity. Two mutations, S258Y (fully mutated) and F174I/L (partially mutated), however, were detected in the production of the RNAi-mediated molecule. An alternative LC-MS approach using dimethyl labeling (i.e., 2CH 2 for rituximab and 2CD 2 for the RNAi-mediated molecule) was developed to additionally compare the two mAbs and confirm the full sequence with the two mutation sites. Furthermore, disulfide linkages were found to be the same for the two antibodies, with a small portion of unpaired cysteines in both products. Disulfides were correctly linked if the samples were prepared at low pH (i.e., enzymatic digestion by pepsin at pH 2); however, trace amounts of scrambling were found by trypsin digestion at pH 6.8, and this scrambling increased significantly at pH 8. Typical modifications, such as pyro-Glu formation at the N-terminus, K clipping at the C-terminus, oxidation at Met, and deamidation at Asn, were also detected with no significant differences between the two products. Using the LC-MS approaches for the comparability study, product integrity with critical structure information was revealed for confirmation of intended purpose (core fucosylation), identification of critical parameters (e.g., sample pH), and correction as needed (amino acid mutation).

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“…The benefits of a transient approach using synthetic siRNA include the ability to outsource production and the fact that multiple siRNA constructs can be generated in a fraction of the time taken to generate constructs in plasmids. Furthermore, synthetic siRNA is efficacious, as evidenced by literature examples of Fut8 gene silencing that are sufficient to reduce FUT8 protein expression 25 and yield afucosylated IgGs with increased FCgRIIIa binding and ADCC 26 .…”

Section: Introductionmentioning

confidence: 99%

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Marbiah

Kotidis

Donini

et al. 2022

JoVE

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Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands.However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9.Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laserinduced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays

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Evaluation of exogenous siRNA addition as a metabolic engineering tool for modifying biopharmaceuticals

Cited by 8 publications

References 34 publications

A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells

A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells

Comparability analysis of anti-CD20 commercial (rituximab) and RNAi-mediated fucosylated antibodies by two LC-MS approaches

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

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