Reduction of medium consumption in perfusion mammalian cell cultures using a perfusion rate equivalent concentrated nutrient feed

Media preparation for perfusion cell culture processes contributes significantly to operational costs and the footprint of continuous operations for therapeutic protein manufacturing. In this study, definitions are given for the use of a perfusion equivalent nutrient feed stream which, when used in combination with basal perfusion medium, supplements the culture with targeted compounds and increases the medium depth. Definitions to compare medium and feed depth are given in this article. Using a concentrated nutrient feed, a 1.8-fold medium consumption (MC) decrease and a 1.67-fold increase in volumetric productivity (PR) were achieved compared to the initial condition. Later, this strategy was used to push cell densities above 100 × 10⁶ cells/ml while using a perfusion rate below 2 RV/day. In this example, MC was also decreased 1.8-fold compared to the initial condition, but due to the higher cell density, PR was increased 3.1-fold and to an average PR value of 1.36 g L⁻¹ day⁻¹ during a short stable phase, and versus 0.46 g L⁻¹ day⁻¹ in the initial condition. Overall, the performance improvements were aligned with the given definitions. This multiple feeding strategy can be applied to gain some flexibility during process development and also in a manufacturing set-up to enable better control on nutrient addition.     

Integration of ion exchange resin materials for a downstream-processing approach of an imine reductase-catalyzed reaction

In this study, an ion exchange resin-based downstream-processing concept for imine reductase (IRED)-catalyzed reactions was investigated. As a model reaction, 2-methylpyrroline was converted to its corresponding product (S)-2-methylpyrrolidine with >99% of conversion by the (S)-selective IRED from Paenibacillus elgii B69. Under optimized reaction conditions full conversion was achieved using a substrate concentration of 150 and 500 mmol/L of d -glucose. Seven commercially available cation- and anion-exchange resins were studied with respect to their ability to recover the product from the reaction solution. Without any pretreatment, cation-exchange resins Amberlite IR-120(H), IRN-150, Dowex Monosphere 650C, and Dowex Marathon MSC showed high recovery capacities (up to >90%). A 150-ml preparative scale reaction was performed yielding ~1 g hydrochloride salt product with >99% purity. Any further purification steps, for example, by column chromatography or recrystallization, were not required.     

Meiofauna promotes litter decomposition in stream ecosystems depending on leaf species

Litter decomposition, a fundamental process of nutrient cycling and energy flow in freshwater ecosystems, is driven by a diverse array of decomposers. As an important component of the heterotrophic food web, meiofauna can provide a trophic link between leaf‐associated microbes (i.e., bacteria and fungi)/plant detritus and macroinvertebrates, though their contribution to litter decomposition is not well understood. To investigate the role of different decomposer communities in litter decomposition, especially meiofauna, we compared the litter decomposition of three leaf species with different lignin to nitrogen ratios in litter bags with different mesh sizes (0.05, 0.25, and 2 mm) in a forested stream, in China for 78 days. The meiofauna significantly enhanced the decomposition of leaves of high‐and medium‐ quality, while decreasing (negative effect) or increasing (positive effect) the fungal biomass and diversity. Macrofauna and meiofauna together contributed to the decomposition of low‐quality leaf species. The presence of meiofauna and macrofauna triggered different aspects of the microbial community, with their effects on litter decomposition varying as a function of leaf quality. This study reveals that the meiofauna increased the trophic complexity and modulated their interactions with microbes, highlighting the important yet underestimated role of meiofauna in detritus‐based ecosystems.     

Analysis of chemometric models applied to Raman spectroscopy for monitoring key metabolites of cell culture

The Food and Drug Administration (FDA) initiative of Process Analytical Technology (PAT) encourages the monitoring of biopharmaceutical manufacturing processes by innovative solutions. Raman spectroscopy and the chemometric modeling tool partial least squares (PLS) have been applied to this aim for monitoring cell culture process variables. This study compares the chemometric modeling methods of Support Vector Machine radial (SVMr), Random Forests (RF), and Cubist to the commonly used linear PLS model for predicting cell culture components—glucose, lactate, and ammonia. This research is performed to assess whether the use of PLS as standard practice is justified for chemometric modeling of Raman spectroscopy and cell culture data. Model development data from five small-scale bioreactors (2 × 1 L and 3 × 5 L) using two Chinese hamster ovary (CHO) cell lines were used to predict against a manufacturing scale bioreactor (2,000 L). Analysis demonstrated that Cubist predictive models were better for average performance over PLS, SVMr, and RF for glucose, lactate, and ammonia. The root mean square error of prediction (RMSEP) of Cubist modeling was acceptable for the process concentration ranges of glucose (1.437 mM), lactate (2.0 mM), and ammonia (0.819 mM). Interpretation of variable importance (VI) results theorizes the potential advantages of Cubist modeling in avoiding interference of Raman spectral peaks. Predictors/Raman wavenumbers (cm⁻¹) of interest for individual variables are X1139–X1141 for glucose, X846–X849 for lactate, and X2941–X2943 for ammonia. These results demonstrate that other beneficial chemometric models are available for use in monitoring cell culture with Raman spectroscopy.     

Parameter estimation for discretely observed linear birth‐and‐death processes

Birth‐and‐death processes are widely used to model the development of biological populations. Although they are relatively simple models, their parameters can be challenging to estimate, as the likelihood can become numerically unstable when data arise from the most common sampling schemes, such as annual population censuses. A further difficulty arises when the discrete observations are not equi‐spaced, for example, when census data are unavailable for some years. We present two approaches to estimating the birth, death, and growth rates of a discretely observed linear birth‐and‐death process: via an embedded Galton‐Watson process and by maximizing a saddlepoint approximation to the likelihood. We study asymptotic properties of the estimators, compare them on numerical examples, and apply the methodology to data on monitored populations.     

粪甲虫对绢蒿荒漠春秋两季牛粪分解的影响

为明确绢蒿荒漠草地牛粪的分解规律,分别在5月(春季)和9月(秋季)设置不同堆置时间点(0、7、29、48、58 h),明确粪便理化性质变化规律,同时设置不同开口的网笼(无网笼、上下开口、上开口、全封口),探讨各生态功能类群粪甲虫对粪便分解的影响。结果表明: 春季的粪甲虫种类数显著高于秋季,秋季的粪甲虫数量显著高于春季。春季粪便的水分、全碳、全氮、全磷下降主要集中在0～29 h,堆置29 h时粪便的水分、全碳、全氮、全磷分别降低39.4%、13.9%、32.1%、26.7%。堆置58 h时粪便的中性洗涤纤维和酸性洗涤纤维分别显著降低8.0%和16.0%。秋季粪便的水分、中性洗涤纤维、酸性洗涤纤维在0～7 h内下降速度最快,堆置7 h时分别降低85.6%、10.2%和20.2%。7～58 h内中性洗涤纤维和酸性洗涤纤维上升,58 h时粪便的中性洗涤纤维和酸性洗涤纤维分别升高20.0%和13.7%。全碳、全氮和全磷的分解主要集中在0～29 h内,堆置29 h时分别降低17.5%、55.0%和64.8%。不同开口的网笼有效阻止了相应生态功能类群粪甲虫的进驻,随着粪甲虫生态功能类群的增加,粪便分解速度加快,无网笼状态下的粪便分解速度显著高于其它处理。粪甲虫种类、数量以及粪便堆置时间均显著影响牛粪的分解过程。     

《国家重点保护野生植物名录》调整的必要性、原则和程序

我国是世界上植物多样性最丰富的国家之一, 1999年发布的《国家重点保护野生植物名录》(下称《名录》(第一批))明确了国家重点保护野生植物的范围, 为依法强化保护、规范无序开发利用、提高公众保护意识奠定了基础。20多年来, 我国野生植物多样性保护形势发生了很大变化, 需要对《名录》进行调整。2018年, 国家林业和草原局、农业农村部启动《名录》调整工作, 物种的遴选遵循了5条基本原则和4条补充性原则, 这些原则主要涉及中国珍稀濒危物种, 具有重要经济、文化、科研、生态等价值物种的入选以及部分物种的排除。经国务院批准, 2021年9月7日, 国家林业和草原局、农业农村部发布了调整后的《名录》, 包括真菌类、藻类、苔藓、石松类和蕨类植物、裸子植物和被子植物, 共计约1,101种(455种和40类)野生植物列入其中。本文简要介绍了《名录》调整的必要性、原则和程序及调整后的情况。     

亚热带6个典型树种吸收细根寿命与形态属性格局

根周转是地下生态过程的主要驱动力, 根属性指征了物种生态策略, 根寿命与属性是理解生态系统碳氮循环和群落多样性的关键。目前对亚热带常绿阔叶林根周转等生态过程的直接观测资料缺乏。该研究对中亚热带江西樟树试验林场6个树种吸收细根动态进行了2年观测, 获取了2.8万张微根管照片, 分析了吸收细根寿命年际和季节变化特征及其与根形态属性的关系。结果显示: 1)亚热带6个树种间吸收细根寿命变异为4.6倍, 变异系数可达73%。中值寿命排序为: 红豆杉(Taxus wallichiana)(426天) >复羽叶栾树( Koelreuteria bipinnata)(155天) >竹柏( Nageia nagi)(145天) >樟( Cinnamomum camphora)(126天) >东京樱花( Cerasus yedoensis)(93天) >深山含笑( Michelia maudiae)(92天); 2)树木吸收细根寿命年际、季节变异较大, 可能是适应伏秋旱、雨热不同期、年际变化大的亚热带季风气候的结果; 3)吸收细根寿命与直径呈显著正相关关系, 与比根长呈显著负相关关系, 表明根的构建成本可以在一定程度上预测寿命。这些结果为预测亚热带地下生态过程、揭示亚热带常绿阔叶林碳氮循环、物种共存机制提供依据。     

Determination of protein concentration in downstream biomanufacturing processes by in-line index of refraction

Protein concentration determination is a necessary in-process control for the downstream operations within biomanufacturing. As production transitions from batch mode to an integrated continuous bioprocess paradigm, there is a growing need to move protein concentration quantitation from off-line to in-line analysis. One solution to fulfill this process analytical technology need is an in-line index of refraction (IoR) sensor to measure protein concentration in real time. Here the performance of an IoR sensor is evaluated through a series of experiments to assess linear response, buffer matrix effects, dynamic range, sensor-to-sensor variability, and the limits of detection and quantitation. The performance of the sensor was also tested in two bioprocessing scenarios, ultrafiltration and capture chromatography. The implementation of this in-line IoR sensor for real-time protein concentration analysis and monitoring has the potential to improve continuous bioprocess manufacturing.     

A novel enzymatic process for glycogen production

Two well-established methods to prepare glycogen are available: (1) extraction from natural resources such as shellfish and animal tissues; (2) synthesis from glucose-1-phosphate using two enzymes, α-glucan phosphorylase (EC 2.4.1.1) and branching enzyme (EC 2.4.1.18). We have developed a novel enzymatic process for glycogen production, in which short-chain amylose is first prepared from starch or dextrin by using isoamylase (EC 3.2.1.68), and then branching enzyme and amylomaltase (EC 2.4.1.25) are added to synthesize glycogen. Our enzymatic process, using isoamylase, branching enzyme and amylomaltase, is currently the most efficient for glycogen production. Furthermore, the molecular weight of glycogen is controllable in a range of 3.0×10⁶ to 3.0×10⁷ by adjusting some parameters of the reaction.