Acid-induced aggregation propensity of nivolumab is dependent on the Fc

Nivolumab, an anti-programmed death (PD)1 IgG₄ antibody, has shown notable success as a cancer treatment. Here, we report that nivolumab was susceptible to aggregation during manufacturing, particularly in routine purification steps. Our experimental results showed that exposure to low pH caused aggregation of nivolumab, and the Fc was primarily responsible for an acid-induced unfolding phenomenon. To compare the intrinsic propensity of acid-induced aggregation for other IgGs subclasses, tocilizumab (IgG₁), panitumumab (IgG₂) and atezolizumab (aglyco-IgG₁) were also investigated. The accurate pH threshold of acid-induced aggregation for individual IgG Fc subclasses was identified and ranked as: IgG₁?1?2?4. This result was cross-validated by thermostability and conformation analysis. We also assessed the effect of several protein stabilizers on nivolumab, and found mannitol ameliorated the acid-induced aggregation of the molecule. Our results provide valuable insight into downstream manufacturing process development, especially for immune checkpoint modulating molecules with a human IgG₄ backbone.     

A high‐throughput capillary isoelectric focusing immunoassay for fingerprinting protein sialylation

The serum half‐life, biological activity, and solubility of many recombinant glycoproteins depend on their sialylation. Monitoring glycoprotein sialylation during cell culture manufacturing is, therefore, critical to ensure product efficacy and safety. Here a high‐throughput method for semi‐quantitative fingerprinting of glycoprotein sialylation using capillary isoelectric focusing immunoassay on NanoPro (Protein Simple) platform was developed. The method was specific, sensitive, precise, and robust. It could analyze 2 μL of crude cell culture samples without protein purification, and could automatically analyze from 8 samples in 4 h to 96 samples in 14 h without analyst supervision. Furthermore, its capability to detect various changes in sialylation fingerprints during cell culture manufacturing process was indispensable to ensure process robustness and consistency. Moreover, the changes in the sialylation fingerprints analyzed by this method showed strong correlations with intact mass analysis using liquid chromatography and mass spectrometry. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:235–241, 2016     

GAP promoter‐based fed‐batch production of highly bioactive core streptavidin by Pichia pastoris

Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin‐auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol‐based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost. Moreover, common methanol‐based processes lead to large proportions of biotin‐blocked binding sites of streptavidin due to biotin‐supplemented media. Targeting these problems, this paper provides strategies for the methanol‐free production of highly bioactive core streptavidin by P. pastoris under control of the constitutive GAP promoter. Complex were superior to synthetic production media regarding the proportion of biotin‐blocked streptavidin. The optimized, easily scalable fed‐batch process led to a tetrameric product concentration of up to 4.16 ± 0.11 µM of biotin‐free streptavidin and a productivity of 57.8 nM h^?1 based on constant glucose feeding and a successive shift of temperature and pH throughout the cultivation, surpassing the concentration in un‐optimized conditions by a factor of 3.4. Parameter estimation indicates that the optimized conditions caused a strongly increased accumulation of product at diminishing specific growth rates (μ ≈ D < 0.01 h^?1), supporting the strategy of feeding. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:855–864, 2016     

Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss

Monitoring the physical or chemical properties of cell broths to infer cell status is often challenging due to the complex nature of the broth. Key factors indicative of cell status include cell density, cell viability, product leakage, and DNA release to the fermentation broth. The rapid and accurate prediction of cell status for hosts with intracellular protein products can minimise product loss due to leakage at the onset of cell lysis in fermentation. This article reports the rheological examination of an industrially relevant E. coli fermentation producing antibody fragments (Fab'). Viscosity monitoring showed an increase in viscosity during the exponential phase in relation to the cell density increase, a relatively flat profile in the stationary phase, followed by a rapid increase which correlated well with product loss, DNA release and loss of cell viability. This phenomenon was observed over several fermentations that a 25% increase in broth viscosity (using induction‐point viscosity as a reference) indicated 10% product loss. Our results suggest that viscosity can accurately detect cell lysis and product leakage in postinduction cell cultures, and can identify cell lysis earlier than several other common fermentation monitoring techniques. This work demonstrates the utility of rapidly monitoring the physical properties of fermentation broths, and that viscosity monitoring has the potential to be a tool for process development to determine the optimal harvest time and minimise product loss. © 2016 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers, 32:1069–1076, 2016     

关于（荒野）殖民化

由于人类体验自然的渴望日益增长,在政治和实践层面,在城市中提供接触自然的机会显得越来越必要。关于“城市荒野”的思想和规划旨在提供一种特殊的自然体验。鉴于不同荒野思想之间存在冲突,风景园林师必须设法了解已有的荒野认知及其含义。通过 3 个荒野类别—“未知荒野”“特定荒野”和“过程荒野”,探讨发展千年的荒野理念,并提出“殖民化”（colonisations）概念作为理解荒野理念发展的一个关键。自然过程伴随着动植物对空间的殖民,而人类进入和占有空间的殖民过程则包含生理、心理和精神 3 个层面的内容。空间命名是一种特殊的、具有精神和象征意味的殖民化形式。例如,人类在城市中发现野生植被,称其为“野性自然”或“城市荒野”。然而,如今大多数（尤其官方）的荒野定义中均排除了人类干扰：一旦被殖民,真正的荒野就不复存在。科学研究对自然过程的殖民化已取得很多成果,但对于人类有关自然和荒野的认知和态度了解并不多。对于风景园林师来说,这有助于更好地理解如何“基于自然进行设计和建造”,对创造令人满意的景观也非常重要。探讨与“城市荒野”有关的论述、规划和设计观点及思想。 由于人类体验自然的渴望日益增长,在政治和实践层面,在城市中提供接触自然的机会显得越来越必要。关于“城市荒野”的思想和规划旨在提供一种特殊的自然体验。鉴于不同荒野思想之间存在冲突,风景园林师必须设法了解已有的荒野认知及其含义。通过 3 个荒野类别—“未知荒野”“特定荒野”和“过程荒野”,探讨发展千年的荒野理念,并提出“殖民化”（colonisations）概念作为理解荒野理念发展的一个关键。自然过程伴随着动植物对空间的殖民,而人类进入和占有空间的殖民过程则包含生理、心理和精神 3 个层面的内容。空间命名是一种特殊的、具有精神和象征意味的殖民化形式。例如,人类在城市中发现野生植被,称其为“野性自然”或“城市荒野”。然而,如今大多数（尤其官方）的荒野定义中均排除了人类干扰：一旦被殖民,真正的荒野就不复存在。科学研究对自然过程的殖民化已取得很多成果,但对于人类有关自然和荒野的认知和态度了解并不多。对于风景园林师来说,这有助于更好地理解如何“基于自然进行设计和建造”,对创造令人满意的景观也非常重要。探讨与“城市荒野”有关的论述、规划和设计观点及思想。     

真菌联合稀碱预处理杜仲叶提取杜仲胶的工艺研究

以杜仲干叶为原料,经稀碱和绿色木霉联合处理以除去杜仲表面覆盖的角质层,同时洗脱一些可溶性杂质,并且破坏杜仲叶的细胞壁与纤维结构,再以石油醚作为提取溶剂,得到粗胶再经丙酮浸洗获得杜仲精胶。用0.45%NaOH在75℃下除去杜仲叶表面的角质层,并且同时脱除一些可溶性活性成分(如:多糖、黄酮类、京尼平苷等),之后得杜仲叶干燥,利用绿色木霉去破坏杜仲叶的细胞壁与纤维结构,在85℃条件下以石油醚作为提取溶剂提取杜仲胶。最后,经测得未处理前杜仲叶中的杜仲胶的含量为1.78%,0.45%NaOH稀碱预处理后含量为3.16%,绿色木霉发酵后含量为4.36%。预处理前,有机溶剂一次提取率为0.73%,纯度为83.7%。稀碱与真菌联合预处理后一次提取率为2.85%,纯度为96.6%。本工艺以稀碱与绿色木霉共同作用,减少了有机溶剂的用量,与传统工艺相比,更加的绿色环保,安全有效,为杜仲胶的提取提供了科学有效的依据。     

Mental illness and well‐being: an affect regulation perspective

Mental health crucially depends upon affective states such as emotions, stress responses, impulses and moods. These states shape how we think, feel and behave. Often, they support adaptive functioning. At other times, however, they can become detrimental to mental health via maladaptive affect generation processes and/or maladaptive affect regulation processes. Here, we present an integrative framework for considering the role of affect generation and regulation in mental illness and well‐being. Our model views affect generation as an iterative cycle of attending to, appraising and responding to situations. It views affect regulation as an iterative series of decisions aimed at altering affect generation. Affect regulation decisions include identifying what, if anything, should be changed about affect, selecting where to intervene in the affect generation cycle, choosing how to implement this intervention, and monitoring the regulation attempt to decide whether to maintain, switch or stop it. Difficulties with these decisions, often arising from biased inputs to them, can contribute to manifestations of mental illness such as clinical symptoms, syndromes and disorders. The model has a number of implications for clinical assessment and treatment. Specifically, it offers a common set of concepts for characterizing different affective states; it highlights interactions between affect generation and affect regulation; it identifies assessment and treatment targets among the component processes of affect regulation; and it is applicable to prevention and treatment of mental illness as well as to promotion and restoration of psychological well‐being.