428 research outputs found

    Apply adsorption technology to solve the UV sensor instability of dynamic control on periodic counter current purification system

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    Cell culture media are a source of major nutrients that provide cell growth and synthesis of proteins. Researchers and suppliers use combine media components test continually in an attempt to find more suitable cell culture media for use in continuous cell culture processes. Commercial media components are a black box for downstream purification researchers who cannot predict the effects of their constituents on resin affinity, absorbability, even for interference with UV instrument. To more effectively utilize in dynamic continuous purification system, the loading product percentage of resin absorbability is typically controlled at 40-60% maximum binding capacity. It is often five times more than traditional batch purification. Therefore, the swing of UV percentage caused by color material in the bulk harvest is the key process parameter in the periodic counter current purification system. We compared different commercial quaternary ammonium adsorbent matrix (such as Stabilized Regenerated Cellulose, Agarose and Styrene Divinyl Benzene Copolymer) to observe their effects on the swing of the UV amplitude. The results show that agarose group has better de-coloration efficacy than the other two commercial quaternary ammonium groups. Please click Additional Files below to see the full abstract

    THE CHANGE OF KNEE KINEMATICS AFTER ANTERIOR CRUCIATE LIGAMENT DEFICIENCY AND RECONSTRUCTION DURING LANDING

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    The purpose of this study were to evaluate the different of knee kinematics analysis after ACLD and ACLR during landing performances. The participants were instructed to finish counter moment jump (CMJ) with arms free 5 times as hard as possible with Vicon motion system and two force platforms. The ACLD showed a significant less knee flexion degree at the peak vertical GRF compared with others. Our founding was similar to the present studies; the impulse during landing among three groups was almost the same, but the RF EMG showed lower after two ACL groups, especially in ACLD

    Maintaining CD4/CD8 ratio and Th1-CTL subsets of chimeric antigen receptor (CAR)-T cells in serum-free culture conditions

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    Chimeric antigen receptor (CAR) T cells therapy is a promising strategy that significantly controlled the progress of cancer diseases. CAR-T cells could kill cancer cells through cellular immune response; therefore, CD8+ cytotoxic T cells are critical for CAR-T cell therapy. However, recent papers reported that CD4+ T helper cells were important for the response and maintenance of CAR-T cells in vivo. Here, we developed a serum-free CAR-T cell preparation process that maintained the T cell population and controlled the T cell subsets. The CD4+ and CD8+ T cell population in CAR-T cells were maintained at averagely 59.4 % and 34.6%, and the major T cell subsets were Th1 cells and cytotoxic T lymphocytes (CTLs), implying the potentially high cellular immune response. To verifying whether the prepared CAR-T cells were exhausted, the expression of several immune checkpoint markers was determined. Of interest, only less than 20% of CAR-T cells at endpoint were PD-1+ or CTLA4+, but more than 40% of CAR-T cells at the endpoint were TIM-3+, implying most CAR-T cells were not exhausted. These CAR-T cells produced more than 1 ng/mL of IFN-γ in the response to the antigen. Altogether, CAR-T cells could be prepared in our serum-free process in the controlling of T cell subsets, leading to potential high therapeutic potency. Please click Additional Files below to see the full abstract

    Continuous purification of monoclonal antibody using periodic counter-current chromatography

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    Integrated and continuous processing of antibody drugs offers several advantages over traditional batch processing in the biotechnology industry. The flexibility of periodic counter-current (PCC) design is performed in the selection of residence time and column numbers on the capture process. In this study, we investigated the association of residence time and product recovery in the downstream PCC purification. A practical operation of PCC as a continuous capture purification step has been applied to 5L perfusion culture, 5L concentrated fed-batch culture, and 50L fed-bath culture. Using an empirical model for the protein breakthrough curve, residence time (RT) was evaluated and the loading flow rate was adjusted to achieve a target RT of 2.25 minutes for monoclonal antibody (mAb). The sample load volume for each column switching was set on 50 and 58% breakthrough curves, mAb recovery was 88 .4% and 88.9%, and buffer consumption was decreased to under half that of the batch process. Overall, more than 40 grams of purified antibody is obtained in 24 hours using a PCC purification system. Comparison of qualities of mAb analyzed by UPLC and reverse phase chromatography show that glycan profiles and purity are quite similar between antibodies obtained from PCC and batch purification, whereas the acidic variants of mAb purified by PCC is higher than that purified by batch mode. The advantages of a continuous downstream capture step are highlighted for our case study in comparison with the existing batch chromatography processes

    In vitro high expansion of chimeric antigen receptor (CAR)-T cells in serum-free process conditions

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    Manufacturing process is an important and complex factor for preparing chimeric antigen receptor (CAR) T cells for therapy. Although serum was widely applied in the culture or expansion of T cells, the quality of serum could be varied from batch to batch, leading to the variation of T cell expansion and quality. In addition, the safety of pathogens from serum and Chemistry, Manufacturing, and Control (CMC) were required to be considered. To overcome the disadvantages of serum application in T cell culture, serum-free and xeno-free culture conditions were required. We intended to develop a rapid serum-free culture condition for the expansion of immune T cells ex vivo. In our optimized serum-free condition, CAR-T cells could be expanded to about 100-200 times to the initial cell number after 6-day culture and the cell viability of all specimens was above 98%. Of interest, the percentage of CAR+ population in all specimens was increases, and the T cell pollutions could be maintained at averagely about 35-40% of CD8+ T cells and averagely about 50-55% of CD4+ T cells after culture. Taken together, our conditions could be applied in the expansion of CAR-T cells for cell therapy to support the minimum requirement of blood or cell samples from patients and to maintain the T cell population. Please click Additional Files below to see the full abstract

    Fidelity-Enriched Contrastive Search: Reconciling the Faithfulness-Diversity Trade-Off in Text Generation

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    In this paper, we address the hallucination problem commonly found in natural language generation tasks. Language models often generate fluent and convincing content but can lack consistency with the provided source, resulting in potential inaccuracies. We propose a new decoding method called Fidelity-Enriched Contrastive Search (FECS), which augments the contrastive search framework with context-aware regularization terms. FECS promotes tokens that are semantically similar to the provided source while penalizing repetitiveness in the generated text. We demonstrate its effectiveness across two tasks prone to hallucination: abstractive summarization and dialogue generation. Results show that FECS consistently enhances faithfulness across various language model sizes while maintaining output diversity comparable to well-performing decoding algorithms.Comment: Accepted as a short paper at EMNLP 202

    Therapeutic protein expression platform of microbial system

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    A number of expression systems have been developed for the production of pharmaceutical products. Pichia pastoris and Escherichia coli expression system operate in our lab and express antibody fragment (scFv), cytokine, protein base adjuvant and vaccine and process enzyme. The expression platform are consisted of three part, first is strain generation , the second is fermentation process development in 250 ml fermentor and the last is process scale-up to 5 litter fermentor. Please click Additional Files below to see the full abstract

    The microbial antibodies secretion expression platform with scale down fermentors

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    Therapeutic antibodies have become one of the most effective therapeutics for human diseases such as cancer, inflammation and viral infection. The production of antibody-based drugs using microbial expression systems is more cost effective with ease of gene manipulation compared to mammalian expression systems. In our team, antibody fragments (ex: BsAb, scFv and Fab) were produced from methylotrophic yeast Pichia pastoris secretion expression system with the AOX1 as driven promoter or E. coli secretion expression system. To achieve high production yield for both system, we investigated fermentation parameter such as base medium, induction medium, induction condition, feeding strategy and pH. For the 250 ml fermentor Pichia system, the nitrogen have been add into glycerol fed medium and/or methanol induction medium and also compared base-medium, buffered glycerol-complex medium (BMGY) and basal salt medium (BS). The highest scFv production was yielded from the basal salt medium as base medium, glycerol fed medium plus nitrogen and multiple carbon source methanol induction medium. This process can yielded over 500 mg/L scFv. After scale-up from 250 ml fermentor to 5L fermentor, the methanol fed-back control system also applied on the 5 L fermentor, can achieve 1.7 g/L scFv in 5 days. The E. coli expression process has passed through screening for high production yield clones in 2 ml deep-well then confirmed by using 250 ml flask scale. Feeding medium, DO, pH etc, parameters were investigated by parallel 250 ml-fermenter. The parameters from 250 ml fermentor were validated by using 5 L fermenter. Under this scale-up procedure, the antibody Fab was 100 folds production yield, production deep well stage at 1 mg/L, production from 250 ml fermentor stage is 50-100 mg/L and production 5 L fermentor stage is over 35-90 mg/L. Although different antibodies will result in different production yield, building a reliable platform to predict production yield from antibody cell clones under deep well and shake flask stage serves a good scale-down model for future scale-up prediction

    Gram Level scFv expression platform of Pichia pastoris

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    The methylotrophic yeast Pichia pastoris secretion expression system has been developed for the antibody fragments (scFv) production platform. The platform includes three technology platforms, the first one is strain generation, the second is fermentation process development in 250 ml fermentor and the last is process scale up to 5 L. A recombinant scFv went through clone generation, include signal peptide tool box, normally yield 2.5 mg/L titer in deep well. Through the fermentation process development of induction medium composition and feeding strategy by Eppendorf Dasgip parallel 250 ml mini fermentor. During induction step, feeding 100% methanol as induction medium can only produce less than 50 mg/L scFv while feeding methanol-sorbitol mixture can significant increase the production yield to 306 mg/L in five days, about 6-folds increase in productivity. With the supply of additional nitrogen source during glycerol feeding step or at induction step, higher scFv production with 510 mg/L can be achieved. Thus, following the medium composition optimization, the production titer was improved 10 folds in 250 ml mini-fermentor stage. Moreover, when we switched the induction medium feeding strategy from DO-stat to the stepwise feeding, the titer increased form 510 mg/L to ~1000 mg/L and yielded another 2- folds improvement. During medium composition and feeding strategy optimization at 250 ml mini fermentor scale, the production titer could increase 20 folds. Overall, the production titer increased 400 folds from cell line generation to 250 ml fermentation parameter optimization. Furthermore, the process parameter can be scale-up to 5 L fernentor achieving \u3e 1 g/L. Recent progress to include BIP in the expression vector gave at least 2 fold improvement in scFv titer in shake flask, the new clone will be optimized in our established 250 ml and 5 L fermentation platform. Please click Additional Files below to see the full abstract
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