(2015) Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells, mAbs, 7:2, 403-412, DOI: 10.1080/19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Abbreviations: CHO, Chinese hamster ovary; HC, heavy chain; LC, light chain; mAb, monoclonal antibody; F2A, 2A peptide derived from the foot-and-mouth disease virus; E2A, 2A peptide derived from the equine rhinitis virus; P2A, 2A peptide derived from the porcine teschovirus-1; T2A, 2A peptide derived from the Thosea asigna virus; GF2A, F2A with the GSG linker; GE2A, E2A with the GSG linker; GP2A, P2A with the GSG linker; GT2A, T2A with the GSG linker; IgG, immunoglobulin G; IRES, internal ribosome entry site; G, glycine; P, proline; K, lysine; MTX, methotrexate; SEC, size exclusion chromatography; MS, mass spectrometry; PFM, protein-free medium; HT, hypoxanthine and thymine; GFP, green fluorescence protein; PVDF, polyvinylidene difluorideLinking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing.

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The functionalization methods of materials based on bacterial cellulose (BC) mainly focus on the chemical modification or physical coating of fermentation products, which may cause several problems, such as environment pollution, low reaction efficiency and easy loss of functional moieties during application. Here, we develop a modification method utilizing the in situ microbial fermentation method combined with 6-carboxyfluorescein-modified glucose (6CF-Glc) as a substrate using Komagataeibacter sucrofermentans to produce functional BC with a nonnatural characteristic fluorescence. Our results indicate that the microbial synthesis method is more efficient, controllable and environmentally friendly than traditional modification methods. Therefore, this work confirms that BC can be functionalized by using a microbial synthesis system with functionalized glucose, which provides insights not only for the functionalization of BC but also for the in situ synthesis of other functional materials through microbial synthetic systems.

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This special issue includes eight original works that detail the further developments of ELMs in theories, applications, and hardware implementation. In "Representational Learning with ELMs for Big Data," Liyanaarachchi Lekamalage Chamara Kasun, Hongming Zhou, Guang-Bin Huang, and Chi Man Vong propose using the ELM as an auto-encoder for learning feature representations using singular values. In "A Secure and Practical Mechanism for Outsourcing ELMs in Cloud Computing," Jiarun Lin, Jianping Yin, Zhiping Cai, Qiang Liu, Kuan Li, and Victor C.M. Leung propose a method for handling large data applications by outsourcing to the cloud that would dramatically reduce ELM training time. In "ELM-Guided Memetic Computation for Vehicle Routing," Liang Feng, Yew-Soon Ong, and Meng-Hiot Lim consider the ELM as an engine for automating the encapsulation of knowledge memes from past problem-solving experiences. In "ELMVIS: A Nonlinear Visualization Technique Using Random Permutations and ELMs," Anton Akusok, Amaury Lendasse, Rui Nian, and Yoan Miche propose an ELM method for data visualization based on random permutations to map original data and their corresponding visualization points. In "Combining ELMs with Random Projections," Paolo Gastaldo, Rodolfo Zunino, Erik Cambria, and Sergio Decherchi analyze the relationships between ELM feature-mapping schemas and the paradigm of random projections. In "Reduced ELMs for Causal Relation Extraction from Unstructured Text," Xuefeng Yang and Kezhi Mao propose combining ELMs with neuron selection to optimize the neural network architecture and improve the ELM ensemble's computational efficiency. In "A System for Signature Verification Based on Horizontal and Vertical Components in Hand Gestures," Beom-Seok Oh, Jehyoung Jeon, Kar-Ann Toh, Andrew Beng Jin Teoh, and Jaihie Kim propose a novel paradigm for hand signature biometry- for touchless applications without the need for handheld devices. Finally, in "An Adaptive and Iterative Online Sequential ELM-Based Multi-Degree-of-Freedom Gesture Recognition System," Hanchao Yu, Yiqiang Chen, Junfa Liu, and Guang-Bin Huang propose an online sequential ELM-based efficient gesture recognition algorithm for touchless human-machine interaction

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Exposure of three native IgG1 monoclonal antibodies to 100mM acetate, pH 3.5 had no significant effect on their hydrodynamic size (11.5±0.5nm), while elution from protein A with the same buffer created a conformation of 5.5±1.0nm. Formation of the reduced-size conformation was preceded by the known destabilization of the second constant domain of the heavy chain (Cγ2) by contact with protein A, then compounded by exposure to low pH, creating extended flexibility in the hinge-Cγ2 region and allowing the Fab region to fold over the Fc region. The reduced-size conformation was necessary for complete elution. It persisted unchanged for at least 7 days under elution conditions. Physiological conditions restored native size, and it was maintained on re-exposure to 100mM acetate, pH 3.5. Protein A-mediated destabilization and subsequent restoration of native size did not create aggregates, but the reduced-size conformation was more susceptible to aggregation by secondary stress than native antibody. Protein A-mediated formation of the reduced-size conformation is probably universal during purification of human IgG1 antibodies, and may occur with other subclasses and IgG from other species, as well as Fc-fusion proteins.