自体CIK细胞对化疗后晚期乳腺癌患者影响的临床对照研究

目的:评估自体CIK细胞治疗对改善化疗后晚期乳腺癌患者机体免疫力及生活质量的疗效及安全性。方法:通过开放的、非随机同期对照研究,比较自体CIK细胞治疗与一般对症治疗对化疗后晚期乳腺癌患者外周血T淋巴细胞亚群的影响,运用E-ORTC QLQ-C30生活质量测评量表分析CIK治疗前后患者生活质量改变,观察不良反应。结果:CIK治疗对化疗后晚期乳腺癌患者外周血CD3+、CD3+CD4+、CD3+CD56+T细胞亚群的升高作用优于一般对症治疗,自体CIK细胞治疗后患者临床症状、躯体功能及总健康状况较治疗前有明显改善,无严重不良反应。结论:自体CIK细胞对改善化疗后晚期乳腺癌患者机体免疫力及生活质量有较好疗效及安全性。     

我国生物医药产业现状及区域化发展战略

正当前,生物科技不断取得新的重大突破,精准医学模式正在推动药物研发革命,基因技术和细胞工程为疾病治疗带来革命性转变,医疗器械开始走向智能化、网络化、标准化,提供了新型诊疗手段,合同研发(CRO)、合同生产(CMO)、第三方检测、健康管理等新兴生物医药服务业态不断兴起和壮大,生物合成等新技术产业化进程进一步加快,这将从根本上改变生物产业的发展模式,为生物经济繁荣发展带来无限想象空间。从产业角度来看,全球生物     

细胞免疫治疗载体技术的现状与展望

近年我国细胞免疫治疗发展迅速,从零基础直追国际前沿水平。在细胞免疫疗法蓬勃发展的背后,将基因导入靶细胞并使其进行表达的基因递送载体技术的支持不可或缺。如何更加安全高效地进行基因转递也是困扰行业发展的重要瓶颈之一。通过总结目前细胞治疗领域主要应用的载体技术发展现状,并对比已经上市产品的工业化生产流程,以期为后续载体技术的发展提供参考。     

皮质酮诱导的PC12细胞梯度应激损伤模型的构建及评价

目的:建立皮质酮诱导的PC12细胞梯度应激损伤模型,为细胞应激水平的评估和细胞应激损伤调控研究提供实验基础和对象。方法:通过检测不同浓度皮质酮(0～1 000μmol/L)在经过不同干预时间(8～48 h)后PC12细胞活力,观察皮质酮对细胞活力的影响,筛选最佳干预条件的细胞模型。分光光度法和微量法检测细胞模型的关键应激指标(MDA、SOD、NADH、LDH),对模型进行评价。结果:当皮质酮浓度在200μmol/L以下且干预时间为12 h时,细胞活力在半数失活率以下,可减少各组由于细胞活力下降而产生的混杂因素。与空白对照组比较,皮质酮浓度依赖性地升高模型组的MDA、NADH和LDH水平,降低SOD水平(P<0.01),符合梯度应激模型的构建要求。结论:成功建立了PC12细胞梯度应激损伤模型,在干预时间为12 h的情况下,干预浓度为0μmol/L、25μmol/L、50μmol/L、100μmol/L、150μmol/L、200μmol/L,使得细胞模型应激损伤程度梯度增加,可作为开展细胞应激损伤评估及调控实验的基础和对象。     

纳米铂黑作为电极镀层的细胞毒性评价

目的:探讨将纳米铂黑作为电极镀层的细胞毒性,初步评估铂黑电镀电极长期植入生物体内的安全性。方法:分别改变混悬液浓度(0.1 mg、0.2 mg、0.3 mg/m L铂黑混悬液)对L-929成纤维细胞培养24 h、48 h、72 h,显微镜下观察细胞形态变化并通过MTS法计算细胞相对增殖率,对细胞毒性进行评级。结果:0.2 mg/m L、0.3 mg/m L的铂黑混悬液中L-929成纤维细胞变成圆形、胞核固缩,细胞稀少,贴壁差;在0.1 mg/m L的铂黑混悬液中上述细胞形态变化则较轻微。0.1 mg/m L的铂黑混悬液细胞毒性为0~2级,且随时间延长而毒性逐渐减弱并呈现出无细胞毒性;0.2 mg/m L、0.3 mg/m L的铂黑混悬液细胞毒性为3~4级,不随时间变化。结论:纳米铂黑其质量体积浓度低于0.1 mg/m L时具有较好的生物相容性。     

SARS-CoV-2入侵细胞的研究进展

新型冠状病毒(SARS-CoV-2)感染引发的肺炎疫情已蔓延全球,尽快认清病毒感染规律和致病机制是做好疫情防控的基础。SARS-CoV-2表面的刺突蛋白(Spike,S)识别靶细胞受体并与之结合,诱导病毒与细胞的膜融合,是病毒侵入宿主细胞的第一步,也是预防和治疗病毒感染的关键靶点。大量研究揭示了病毒进入细胞的分子机制,本文将主要对SARS-CoV-2入侵细胞的研究成果进行总结,并简要叙述以该环节为靶点的药物和疫苗研发现状。     

DCs-CIK细胞免疫治疗联合化疗治疗晚期非小细胞肺癌的疗效观察

目的:探讨树突状细胞(DCs)和细胞因子诱导的杀伤(CIK)细胞免疫治疗联合化疗对晚期非小细胞肺癌患者的治疗效果。方法:将我院2012年2月到2014年2月就诊的72例晚期非小细胞肺癌患者随机分为对照组(n=36,单纯化疗组)和实验组(n=36,DCs-CIK细胞免疫联合化疗组)。比较两组患者治疗后的疗效、治疗前后免疫功能,并运用Kamofsky(KPS)评分来评估两组患者治疗后生活质量的改善情况。结果:实验组的疾病控制率(DCR)77.78%显著高于对照组的52.78%(P0.05)。治疗后实验组患者外周血CD3+、CD8+及NK细胞所占的比值较治疗前均上升显著(P0.05);治疗后对照组患者外周血CD3+、CD8+及NK细胞所占的比值较治疗前下降显著(P0.05)。治疗后实验组KPS评分提高率明显高于对照组(P0.05)。结论:DCs-CIK细胞免疫联合化疗能够提高晚期非小细胞肺癌患者的DCR,且显著改善患者的免疫功能和生活质量。     

灵武长枣果实维管束韧皮部及周围细胞的超微结构特征

为了探讨灵武长枣果实光合同化物韧皮部卸载和运输的途径,该研究采用透射电镜技术,对不同发育时期灵武长枣果实维管束韧皮部及其周围薄壁细胞的超微结构特征进行了分析.结果表明:筛管/伴胞复合体及其周围韧皮薄壁细胞间在果实膨大前期富含胞间连丝,而韧皮薄壁细胞与周围库细胞以及相邻库细胞间几乎不存在胞间连丝,形成共质体隔离;筛管/伴...     

敲除Usp13促进棕榈酸诱导的小鼠肝实质细胞凋亡*

目的: 研究去泛素化酶Usp13对棕榈酸诱导的肝实质细胞凋亡的影响,并对其机制进行初步探究。方法: 两步胶原酶灌注法分离小鼠原代肝实质细胞并采用棕榈酸处理;甘油三酯酶法及油红O染色检测肝实质细胞中脂质的累积;MTS和LDH试剂盒检测细胞活力;流式细胞术检测细胞凋亡;qPCR检测凋亡相关基因的表达以及免疫印迹法检测Caspase-3的活化。结果: 敲除Usp13基因并不影响棕榈酸诱导下肝实质细胞的甘油三酯累积,但导致细胞损伤加重,凋亡增加。机制研究显示,敲除Usp13促使肝实质细胞中抗凋亡基因Bcl-2、Bcl-xl表达下调,促凋亡基因Bid、p53表达上调,以及Caspase-3活化增强。结论: 初步揭示了Usp13调控棕榈酸诱导下肝实质细胞凋亡的机制,为深入研究Usp13调控非酒精性脂肪性肝炎的发生、发展奠定了基础。     

中枢神经系统神经胶质细胞对神经病理性疼痛的调节作用

神经病理性疼痛是一种临床的常见疾病,严重影响了患者及家属的生活质量,给社会带来了沉重的负担。神经病理性疼痛的发病机制及有效治疗仍在探索中。中枢神经系统内有三种胶质细胞,包括小胶质细胞、星形胶质细胞以及少突胶质细胞。近来有研究发现,这三种胶质细胞可通过活化、产生和释放细胞因子等途径参与神经病理性疼痛的调节。探索神经胶质细胞的多种复杂功能或作用机制来充分认识胶质细胞的特点,为今后神经病理性疼痛的临床治疗提供新的思路。本文通过研究小胶质细胞、星形胶质细胞以及少突胶质细胞的特点及其对神经病理性疼痛的影响,并分析中枢神经系统胶质细胞与疼痛治疗之间的相关性,旨在总结神经病理性疼痛的发生和发展过程中小胶质细胞、星状胶质细胞及少突胶质细胞的调节作用。     

Biotech Outsourcing Strategies cmc—Biologics stream: June 17, 2010, Copenhagen,Denmark

Now in its third year, the Biotech Outsourcing Strategies (BOS) meeting organized by Bio2Business took place at the Søhuset Conference Centre in Hørsholm, Copenhagen. The focus of this year''s event was the demanding and challenging area of chemistry, manufacturing and controls (CMC), and the meeting provided ample opportunity for lively discussion of the key issues surrounding this area. New for the 2010 conference, a biologics-focused lecture stream ran in parallel to the established small molecule stream. Both streams boasted a distinguished panel of keynote speakers who discussed all aspects of CMC from early stage scale-up through late stage clinical development. In addition to the keynote speakers, selected contract research organizations (CROs) gave short presentations on the solutions that they could provide to some of the challenges facing CMC. The meeting attracted more than 150 delegates from leading drug development companies and CRO service providers, and greatly facilitated the forging of new working relationships through pre-arranged one-to-one meetings. Moreover, exhibitions from event sponsors and considerable scheduled networking time over lunch and evening receptions further enhanced the highly productive and interactive nature of the meeting.Key words: outsourcing, chemistry manufacturing and controls, biopharmaceutical developments, contract manufacturingDr. Anne Bondgaard Tolstup (Symphogen) presented an overview of the key challenges for small companies involved in biopharmaceutical up-stream process (USP) development and good manufacturing practice (GMP). Founded in 2000, Symphogen focuses on cell line development and manufacturing of recombinant antibodies including polyclonal antibodies, a new class of therapeutics that Symphogen has brought into clinical development. Their therapeutic areas of interest are cancer, infectious diseases and immunoglobulin replacement therapy because there are many well-understood and novel targets for which new monoclonal antibody (mAb) products can be developed.Central to Dr. Tolstup''s presentation was a critical analysis of their experiences in contract manufacturing organization (CMO) outsourcing of three recombinant antibodies developed in-house. Symphogen''s first outsourcing experience came in 2004, which was fairly early in the life of the company, with the development of Sym001. Sym001 presented significant challenges for outsourcing because it was a new type of polyclonal antibody product comprising 25 recombinant antibodies. The challenge presented by the complexity of Sym001 was further complicated by the limited in-house experience in CMC development and a lack of equipment for USP development at Symphogen. For their USP development strategy, they wanted to employ a single batch manufacturing process that was novel at the time. Starting with 25 individual cell lines under GMP conditions, they expanded the cell lines, mixed them together and generated a polyclonal master cell bank. This was further expanded to give the polyclonal working cell bank that could then be used in USP manufacturing similar to those used for mAb production. To cope with the challenges faced with outsourcing such a novel production method, Symphogen formed an in-house CMC and regulatory team, sought advice from several experienced consultants and conducted extensive research into CMOs and regulatory agencies. After evaluation of several European CMOs, they formed a collaboration with Biovitrum (Stockholm, Sweden). The key criterion for this selection was a sense of strong commitment from the Biovitrum business development team to understand and accommodate customer demands during negotiation of technical work. Dr. Tolstup detailed the working infrastructure of the collaboration, stressing the importance of regular face-to-face meetings between the project teams, and the necessity for a joint steering team to mediate disagreements, thus keeping disputes out of the scientific groups. The collaboration was successful, and resulted in development of a consistent manufacturing process, and release and characterization tools to assess polyclonality. In 2007, Sym001 entered clinical trials and it is currently in Phase 2.In the case of Sym002, a smallpox drug comprising 26 antibodies that is being developed with funding from the United States (US) government, Dr. Tolstup discussed the difficulties that can be encountered when working with CMOs. She highlighted lack of face-to-face meetings, staff inexperience and inexperienced project management teams as less than ideal foundations for a mutually beneficial working relationship. Despite issues surrounding the collaboration, it resulted in two successful batch scale-ups of Sym002.The third CMO experience at Symphogen came in the manufacturing of Sym004, which comprises two chimeric IgG1 antibodies that target epidermal growth factor receptor domain III at uniquely positioned, non-overlapping epitopes. The manufacturing strategy for Sym004 included separate USP and down-stream process (DSP), preparation of drug substance for the two mAbs comprising Sym004, followed by formulation of the mAbs into one drug product by a 1:1 mix. They chose CMC biologics (Copenhagen, Denmark) to collaborate with because their technical equipment fit well with in-house technology at Symphogen. The scientific teams worked well together, leading to successful technology transfer and a robust and scalable USP, and Phase 1 clinical studies are currently underway in the US. Dr. Tolstup concluded by summarizing the general learning points from CMO outsourcing at Symphogen by stating that good communication, flexibility and open-mindedness are key for successful CMO collaborations.Dr. Andreas Castan showcased the implementation of quality by design (QbD) in process development at Biovitrum. Although not an entirely new concept, QbD aims to design and build quality into product and manufacturing processes, instead of simply testing for quality after production. Regulatory agencies require monitoring and process robustness in order to reduce variability of manufacturing, and this is achieved at Biovitrum by the integration of process analytical technology (PAT) into QbD. PAT is a system for designing, analyzing and controlling manufacturing through measurements of critical quality and performance attributes, with the goal of ensuring final product quality. Dr. Castan stressed how incorporation of PAT in QbD can result in reduced variation and increased process understanding, and greatly facilitates technology transfer and scale-up. Linking performance of the manufacturing process to safety and efficacy of the product was discussed, with an emphasis on the need to define the critical quality attributes needed for assessment of the impact that the manufacturing process can have on product safety and efficacy. Dr. Castan indicated that quality attributes are usually assessed by conducting release tests used to assess a small subset of product characteristics, followed by more extensive characterization. By adopting a QbD approach the focus is much more on the process that allows broader assessment of the quality attributes, rather than on analyzing a small representative sample post-manufacture. Defining the relevant critical quality attributes of a product, however, can prove challenging, and Dr. Castan discussed the need for continual reassessment of quality attributes based upon structure-function relationships identified from biological characterization studies. Additionally, he pointed out the requirement for not just one, but a combination of different quality attributes.In a detailed case study, the process whereby Biovitrum defined the design space for one of their projects was explored. The USP comprised a mammalian cell culture fed-batch process, and the DSP consisted of three chromatographic steps, one virus reduction step and one ultrafiltration/diafiltration step, followed by an extensive analytical package to assess the critical quality attributes. Initial risk assessment starts with the manufacturing process and evaluates the operating parameters for each step. Each step is then rated and assigned a risk priority number and further scientific discussion determines which parameters are most likely to have an impact on the quality attributes, and therefore require further characterization. From the process risk assessment, Biovitrum then selected the best design for the project and established that a large number of analytical methods were required.At this point, Dr. Castan touched on experimental design, specifically the difference between running one factor at a time versus a sum of experiments design, which offers a more comprehensive and structured approach to exploration of design space. The characterization of the USP bioreactor step and the DSP capture step using the more structured design of experiments approach was discussed. From the failure mode and effects analysis, operational parameters were identified and augmented with axial points and repeats resulting in a total number of experiments required. The experimental results were then used to assess the reproducibility of the center points of one parameter, which then generated a reliable model necessary for plotting contour charts. With the design space established, it was then possible to identify the desirable product operating ranges.Some of the unique challenges and strategies for outsourcing biologics were evaluated by Dr. Torben Lund-Hansen (Lund-Hansen Consulting ApS). The talk began with an overview of how biologics differ from small molecules specifically with respect to their size, complex and often relatively undefined mechanism of activity, and requirement for post-translational modification. In addition, the source material for biologics can potentially lead to the transmission of adventitious agents that can be hard to identify and may not be easily measurable. As a result, there is a need for substantial in-process control and validation. Dr. Lund-Hansen highlighted how the bio-friendly nature of the manufacturing process aids the susceptibility of the process to microbial contamination, but that it is simply not possible to employ aseptic processing throughout, especially in the drug substance purification.In discussing the pre-clinical attributes of biologics, the difficulties with establishing the pharmacokinetics (PK) of biologics such as antibodies were raised by Dr. Lund-Hansen. While it is possible to measure the antibody in humans or animals for three to four weeks, the biological function may last for many more months, suggesting that PK may not be the most suitable method for assessing biologics. Furthermore, all biologics have an immunogenic potential that is obviously desirable in vaccines, but is a serious unwanted effect that can occur in single and chronic dosing of therapeutics. For example, in some cases up to 20–30% of patients may have an immune response to a protein drug, and this response may in turn cause a substantial decrease in the drug''s efficacy. As a consequence, regulatory agencies have issued very strict guidelines for measuring immunogenicity, which should be of utmost consideration when developing biologics.Another challenging aspect of biologics development is that manufacturing facilities as well as the products must meet regulatory agencies'' specifications. Indeed, prior to obtaining a license the manufacturing facility must undergo inspection while being fully operational and manufacturing the complete product for which a biologics license is required. With this as context, Dr. Lund-Hansen discussed the challenges surrounding CMO selection for biologics. He advised choosing a strong and experienced CMO that can take the product all the way to market so that there is no need for technology transfers during the clinical development process. Furthermore, he stressed the need to work very closely with the CMO to understand the way the CMO works, and, crucially, not try to change their methods. The expectations from a customer point of view were discussed with respect to who is ultimately responsible for ensuring that CMC standards are met. Sometimes CMOs do not take responsibility for CMC and it can often be a sticking point in negotiations between customers and CMOs because of the large degree of risk involved.In assessing the interconnecting relationships between CMOs, sponsors and regulatory agencies, Dr. Lund-Hansen pointed out that quite often the sponsors are relatively inexperienced compared with regulatory agencies and CMOs that have worked with multiple sponsors. He called upon the CMO to educate without intimidating the customer, to be transparent in order to generate trust, and to communicate clearly and precisely the CMO strategy and cost. Additionally, the need to offer the customer a choice was raised with respect to the degree of flexibility within the working contract, and he suggested that a stretched time-line should result in a lower price to the customer.The presentation concluded with an evaluation of the huge amount of risk involved in the development of biotechnology products. Two types of risk were identified: the project risk, which should be carried by the customer and the technical risk, which should be shared between the customer and the CMO because the CMO should be in a better position to mitigate technical risk. Despite this, most CMOs prefer not to assume the technical risk, and this can lead to problems in contract negotiation.The next session featured short presentations from three CROs highlighting the technological solutions that they could provide to various CMC functions. Dr. Michael Becker summarized the technologies that Solvias offers in biopharmaceutical analysis. Solvias focuses on ensuring that the analytical technology that they develop for the customer meets deadlines and milestones to enable efficient product development. They provide analytical support from characterization of proteins through quality control methods to support stability studies, comparability studies and method transfer to CMO sites, focusing mainly on drug substance and drug development. Solvias adopted a fit for purpose approach that allows for innovation to meet customer demands, and they also seek to standardize wherever possible. The key technologies they offer are analysis by fragmented antibody capillary electrophoresis, peptide mapping and routine methods for amino acid analysis, and DNA sequencing.Eva Balslev Jørgensen described the biomanufacturing solutions offered by Novozymes BioPharma. In 2007, Novozymes BioPharma created BioBusiness as a division of Novozymes to strengthen the Novozymes brand position outside enzymes and offer value for customers interested in products with non-enzyme activities. They structure their services into three areas: manufacturing technologies, biomedical applications and manufacturing supplements. In manufacturing technologies, they offer a novel yeast expression system and an albumin infusion technology that can be used as a stabilizer for protein-based drugs. For biomedical applications, they developed the Recombumin excipient for vaccine formulation, albucult^®, which is a stabilizing agent and Hyacare^®, which is used for co-formulation in drug delivery. With respect to manufacturing supplements, they offer cell culture ingredients such as recombinant transferrin and recombinant human growth factors.The final CRO presentation was delivered by Dr. Roman Hlodan (Patheon) who discussed points to consider when developing biopharmaceuticals. Dr. Hlodan began with a look at factors affecting the development pathway by discussing business models, resources, drug delivery and expectations of investors. Regarding formulation choices, Dr. Hlodan suggested that perhaps refrigerated or ambient stored solutions offer the best option as they are cheap, easy to transport and carry the potential to be smoothly transferred to a more desirable delivery such as pre-filled syringe. If this type of formulation is not viable for the drug product, frozen solutions, which can minimize risk to product stability or lyophiles, which offer convenient transportation and storage, can be considered. To conclude his talk, Dr. Hlodan discussed some of the challenges that can arise when manufacturing, including drug substance availability, technology transfer and analytical considerations.The afternoon session began with discussion of approaches to biopharmaceutical formulation and development. Dr. Niels Johansen (ALK) noted that the company focuses on development of biopharmaceutical products for the diagnosis, treatment and prevention of allergies. Allergies are an immunological overreaction to allergens that exist in substances such as pollen, animal hair or food, and the prevalence of allergic diseases is increasing. Most treatments involve simple avoidance and symptomatic medicine, i.e., drugs that control symptoms but not the allergy. Allergy vaccinations, which involve injections of controlled doses of purified and standardized allergens extracted from natural allergen sources, are currently the only treatment that can change the course of the disease. Over time, the injections lead to desensitization of the immune system towards the allergen.Traditionally, allergy vaccinations are administered via subcutaneous injections over a period of three years and are used in the treatment of pollen, grass, dust, animal and bee/wasp venom allergies. In 2005 an alternative vaccination method, sublingual immunotherapy (SLIT), was developed. SLIT provides the potential for a more patient-friendly treatment and ALK strives to deliver an allergy vaccine that can provide fast drug release, solid dosage form for sublingual administration and good shelf life.In discussing drug product formulation, Dr. Johansen examined the requirements for compatibility between the complex mixture of proteins that make up the drug substance and the desired patient-friendly sublingual administration route. He also emphasized the importance of having a carefully designed manufacturing process because protein allergens are highly sensitive to heat and mechanical stress during processing. The question of carrying out in-house drug formulation and delivery development versus outsourcing to a CMO was then raised, and the conclusion was that the only viable option available to ALK as a small company was outsourcing to a CMO. Regarding CMO selection, Dr. Johansen emphasized examination of the reputation, available technology, and the size of the CMO with respect to it being able to take the product through development to market, when choosing a CMO. ALK formed a collaboration with Catalent, UK, the developer of Zydis^®, which is a freeze-dried oral solid dosage formulation technology that allows instant dissolution in the mouth and results in immediate release of the drug. Furthermore, the manufacturing processes of Zydis^® were ideal for formulation of proteins, which are generally stable in freeze-dried formulation. In 2006, ALK launched Grazax^®, which is a fast dissolving oral tablet developed using Zydis^® technology for the treatment of grass pollen-induced rhinitis and conjunctivitis.The strategies for creating outsourcing partnerships at Genmab were discussed by Dr Jesper Valbjørn. Genmab develops fully human antibodies for the treatment of cancers, particularly those for which there is an unmet medical need. Currently, the company has 9 Phase 3 studies of ofatumumab (Arzerra) and 2 Phase 3 studies of zalutumumab on-going, and 3 additional antibodies (daratumumab, RG4930, RG1512) in clinical development. In 2009 their first product, ofatumumab, was approved in the US for the treatment of chronic lymphocytic leukemia. Dr. Valbjørn began by examining the process for selecting a CMO. He noted that selection should be based upon the strategy and needs of the project, and he made clear distinctions between a strategic selection process, which is usually a long term, capacity-driven plan adopted by global companies and a tactical process, which is usually project-driven and includes a limited budget. Additionally, he emphasized that the need to plan for the lifetime of a project before seeking a CMO is key for the success of the project. In a more detailed breakdown of the selection process, Dr. Valbjørn described a method whereby a number of selection criteria are established, weighted and then used to screen potential CMOs.Contracting and project definition were touched upon briefly within the context of the overall process that starts with proposal requests, and includes technical and QA site visits with a review of findings and the forging of technical and quality agreements. Dr. Valbjørn stressed the importance of taking adequate time for this process because getting the agreement right from the start results in a win/win for both sides. Once the contract is in place, the next step is to effectively manage interactions with the CMO. At Genmab, this is done by setting well-defined expectations, goals and responsibilities and having alignment of teams across the company and the CMO. Dr. Valbjørn cited communication, including regular face-to-face meetings, as being important to the success of the project. Disputes are dealt with by a contract manger to keep disagreements out of the technical teams, and joint steering meetings occur every six to twelve months to evaluate performance and align expectations.With regard to technology transfer from the company to the CMO, Dr. Valbjørn stated that transparency with transfer development documentation is necessary in order to prevent problems arising at a later date. He also suggested running the process in the CMO''s development laboratory and training the CMO technicians in specialized analytical methods at your own site. The need to be data-driven and not to make assumptions about the knowledge of the CMO was also stressed. Dr. Valbjørn concluded the presentation with an analysis of the manufacturing process with respect to the engineering run and the GMP run. Engineering runs help to test the scale and performance of the process and minimize risk for the GMP run. Many CMOs are reluctant to carry out a GMP run without an initial engineering run; if the engineering run is eliminated, then there is a requirement to know the process and product well, and to communicate your priorities and risk profile/tolerance to the CMO upfront.Dr. Per Edebrink (RecipharmCobra Biologics) discussed finding the optimal characterization strategy for clinical study material. He began by giving a summary of critical quality attributes, looking at what processes can influence them and stressed the need to define them early in order to determine what characterization is required and aid design of the analytical test package. Although, it may not always be possible to know the critical quality attributes early in the pharmaceutical development, it is possible to predict what may be required from the International Conference on Harmonisation (ICH) guidelines for biopharmaceuticals. In particular, Dr. Edebrink highlighted Q6B, which outlines what structural characterization and physicochemical properties are needed. Regarding potential critical quality attributes of mAbs, Dr. Edebrink mentioned a number of different product variations, including Fc glycosylation, fragmentation in the hinge region, disulfide shuffling and lysine truncation. It is possible to identify some of these variations using intact mass protein analysis, and some example spectra were provided, including a reversed-phase high-performance liquid chromatography-ultraviolet (RP-HPLC-UV) mass spectrometry (MS) spectrum of a mAb showing the free light chain, hinge region fragments and the intact IgG.On the topic of glycoproteins, Dr. Edebrink discussed the effect that glycosylation may have on functional activity. Indeed, glycosylation can affect the biological properties of the glycoprotein, resulting in possible immunogenetic effects, and it can also reduce both the in vivo half-life and the shelf life of the glycoprotein. However, there are many ways to measure the extent of glycosylation of a glycoprotein and Dr. Edebrink showed how electrospray mass spectrometry, high performance anion exchange chromatography and matrix-assisted laser desorption/ionization MS can be used to assess glycoprotein glycosylation. It is also possible to test for sialylation, which can affect the half-life or activity of some biologics. LC-MS can be used to analyze the degree of sialylation and for peptide mapping. The final potential critical quality attribute (CQA) examined was deamidation, which can adversely affect activity. Deamidation is the degradation of asparagine and aspartate that proceeds via a succinamide intermediate. It is possible to test for deamidation using a number of different techniques and examples of using isoelectric focusing and peptide mapping coupled to LC-MS were shown. To conclude, Dr. Edebrink reiterated the need to identify potential CQAs as early as possible using the available knowledge, and assess the potential effect on the product. Furthermore, he highlighted peptide mapping by LC-MS as a powerful tool to provide detailed information on the presence and distribution of product variants.To conclude a highly informative and productive meeting, Dr. Jesper-Sonne Johansen (Novo Nordisk) presented a case study on world-class CMC development processes in mammalian, yeast and bacteria cell lines. In 2006, Novo Nordisk initiated a program to improve their CMC processes and set the ambitious goal of doubling the number of new projects in development within five years, which necessitated a doubling of capacity. They began by mapping all the CMC development processes, such as fermentation and analytical processes, and examined the number of people, time and resources allocated to each process, and assessed if there was room for improvement. They also examined how other companies carried out their CMC development and investigated opportunities for improvement and standardization of technologies going into the CMC process.The company then made two major modifications to their processes. First, they changed from continuous CMC development to a two-step development process in which the first step is to develop suitable processes for production of Phase 1 and 2 drug substance, focusing on delivering a high-quality product through a process that might not be optimized. The second step involves development of a robust manufacturing process and enhanced product knowledge. When implementing this new two-step development process, the decision was made to maintain resources present in Novo Nordisk and not to increase outsourcing.The second change that was implemented was the ‘five initiatives’ process. When examining project transfer from research to development and from development to production, they observed that a substantial amount of knowledge was lost. In order to combat this problem, they established technology transfer teams at each handover one year before the transition would occur to ensure efficient transitions. In addition, they set up technology groups that examined the technology used across research, development and production and, where possible, introduced standardized techniques.Regarding the qualitative outcome of these changes, for the two-step development process the result was a short and focused development time with clear goals and agreements on tasks. In the handover process, the transfer teams co-operated successfully and created knowledge across the chain from research to development to production. For the technology standardization, they developed standard guidelines for purifications and analytical methods. The same technology is now used in many projects, which has resulted in predictable outcomes of processes, and has greatly increased process knowledge. Remarkably, when Novo Nordisk reviewed the effect of implementing these approaches in standard development projects over two years, they found that overall resource use was half the previous level. Overall, the overhaul of CMC development at Novo Nordisk resulted in a two-fold increase in productivity between 2006 and 2008, and they were able to successfully double the number of development projects without increased use of resources.     

透明质酸生产菌溶血素S基因缺失突变菌株的构建及其特性北大核心CSCD

【目的】马链球菌兽疫亚种是工业上生产透明质酸的主要菌种,该菌能产生引起宿主细胞溶血的链球菌溶血素S(streptolysin S,SLS)毒素,因而其产品的安全性一直是人们所担心的问题。本实验的目的就是通过基因敲除的方法构建不产SLS的透明质酸生产工程菌,同时探讨溶血素sag A基因缺失对菌株透明质酸合成和其他毒力因子的影响。【方法】利用温度敏感/自杀性质粒p JR700载体系统,构建马链球菌兽疫亚种sag A基因缺失突变株;通过PCR扩增,溶血平板和SLS含量测定等方法确定sag A基因缺失;采用分光光度、SDS-PAGE和细胞毒性试验等分析方法,对野生菌株和sag A基因缺失突变菌株透明质酸含量、透明质酸分子量、溶血素Hylc、透明质酸分解酶、甘油醛-3-磷酸脱氢酶和菌体表面蛋白等相关毒力因子进行对比研究。【结果】获得了透明质酸产量提高30%而溶血活性极低的马链球菌兽疫亚种sag A基因缺失突变株。该突变株与野生菌株相比较,透明质酸分解酶活性增加而透明质酸相对分子量降低,此外,与毒力相关的表面蛋白含量、溶血素Hylc和甘油醛-3-磷酸脱氢酶活性也显著降低。细胞毒性实验结果表明,野生菌株与sag A基因缺失突变菌株的培养物上清液,对细胞活性的影响存在显著差异。【结论】在马链球菌兽疫亚种中sag A不仅是表达溶血素SLS的基因,同时sag A基因对菌株透明质酸合成、透明质酸分解酶、菌体表面蛋白、溶血素Hylc和甘油醛-3-磷酸脱氢酶等都具有调节作用。     

Technological progress and challenges towards cGMP manufacturing of human pluripotent stem cells based therapeutic products for allogeneic and autologous cell therapies

Recent technological advances in the generation, characterization, and bioprocessing of human pluripotent stem cells (hPSCs) have created new hope for their use as a source for production of cell-based therapeutic products. To date, a few clinical trials that have used therapeutic cells derived from hESCs have been approved by the Food and Drug Administration (FDA), but numerous new hPSC-based cell therapy products are under various stages of development in cell therapy-specialized companies and their future market is estimated to be very promising. However, the multitude of critical challenges regarding different aspects of hPSC-based therapeutic product manufacturing and their therapies have made progress for the introduction of new products and clinical applications very slow. These challenges include scientific, technological, clinical, policy, and financial aspects. The technological aspects of manufacturing hPSC-based therapeutic products for allogeneic and autologous cell therapies according to good manufacturing practice (cGMP) quality requirements is one of the most important challenging and emerging topics in the development of new hPSCs for clinical use. In this review, we describe main critical challenges and highlight a series of technological advances in all aspects of hPSC-based therapeutic product manufacturing including clinical grade cell line development, large-scale banking, upstream processing, downstream processing, and quality assessment of final cell therapeutic products that have brought hPSCs closer to clinical application and commercial cGMP manufacturing.     

Capacity planning for batch and perfusion bioprocesses across multiple biopharmaceutical facilities

Production planning for biopharmaceutical portfolios becomes more complex when products switch between fed‐batch and continuous perfusion culture processes. This article describes the development of a discrete‐time mixed integer linear programming (MILP) model to optimize capacity plans for multiple biopharmaceutical products, with either batch or perfusion bioprocesses, across multiple facilities to meet quarterly demands. The model comprised specific features to account for products with fed‐batch or perfusion culture processes such as sequence‐dependent changeover times, continuous culture constraints, and decoupled upstream and downstream operations that permit independent scheduling of each. Strategic inventory levels were accounted for by applying cost penalties when they were not met. A rolling time horizon methodology was utilized in conjunction with the MILP model and was shown to obtain solutions with greater optimality in less computational time than the full‐scale model. The model was applied to an industrial case study to illustrate how the framework aids decisions regarding outsourcing capacity to third party manufacturers or building new facilities. The impact of variations on key parameters such as demand or titres on the optimal production plans and costs was captured. The analysis identified the critical ratio of in‐house to contract manufacturing organization (CMO) manufacturing costs that led the optimization results to favor building a future facility over using a CMO. The tool predicted that if titres were higher than expected then the optimal solution would allocate more production to in‐house facilities, where manufacturing costs were lower. Utilization graphs indicated when capacity expansion should be considered. © 2013 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:594–606, 2014     

两种精原干细胞高效能纯化方法的比较研究*

目的: 比较贴壁分离法和免疫磁珠法纯化小鼠精原干细胞(mSSCs) 的优缺点。方法: 分别选取10只12-15日龄的雄性C57BL/6小鼠,颈椎脱臼法处死,摘取睾丸用酶消化法获得曲细精管单细胞悬液,分别用贴壁分离法和免疫磁珠法从单细胞悬液中分离纯化mSSCs,并针对两种方法在细胞数量、分离效率以及对细胞增殖生长的影响等方面进行比较。结果: 两种纯化方法均可从小鼠曲细精管单细胞悬液中分离纯化得到干细胞,并可在体外培养后呈现出典型的精原干细胞特有的葡萄串状克隆,体外连续培养增殖超3个月。10只小鼠的睾丸经差异贴壁法纯化后可以得到3×10⁵±0.4×10⁵个mSSCs(n=5),细胞回收率(纯化后细胞数/曲细精管单细胞悬液细胞数)为1.5%±0.1%(n=5);经免疫磁珠法可以得到6×10⁵±0.4×10⁵个mSSCs(n=5),细胞回收率为3.0%±0.1%(n=5),免疫磁珠法得到的干细胞数量更高。差异贴壁法得到的干细胞更纯,因为体外培养5 d左右即得到干细胞集落,而免疫磁珠法得到的干细胞则约10 d才可以看到明显的细胞集落,但是两种纯化方法对细胞体外长期增殖生长没有明显的影响。结论: 两种方法均可以纯化得到高质量的mSSCs,,但两种方法各有优缺点。差异贴壁法较免疫磁珠法经济、实用,无需购买专门的设备和抗体磁珠,但获得的细胞数量相对较低,用时也较长。     

山西省健康成年人淋巴细胞亚群正常参考值范围

目的:建立山西省健康成人外周血淋巴细胞亚群的正常参考值范围,为机体免疫状态的分析和肿瘤患者的免疫评估提供理论依据。方法:选取山西省1 238例健康成人体检人群,采用流式细胞术测定外周血淋巴细胞亚群的绝对计数和相对计数。结果:确定了健康成人外周血淋巴细胞表达水平,并发现CD3~+T细胞相对计数和绝对计数、CD4~+T细胞相对计数、CD8~+T细胞相对计数和绝对计数、NK细胞相对计数和绝对计数、CD19细胞相对计数和绝对计数、CD4/CD8比值在不同年龄组间存在显著差异性(P0.05);不同性别之间CD8~+T细胞相对计数、CD4~+T细胞绝对计数和CD19细胞绝对计数无统计学意义,CD3~+T细胞、CD8~+T细胞、NK细胞相对计数和绝对计数、CD4~+T细胞、CD19细胞相对计数均存在显著差异(P0.05)。结论:初步建立了山西省健康成年人外周血淋巴细胞亚群参考值范围,为机体免疫功能的评价和肿瘤免疫治疗、诊断提供了参考依据。     

Twenty-Four-Hour Variations in the Sensitivity of Rat Aorta to Vasoactive Agents

The presence of time-dependent variations in the in vitro sensitivity of aorta preparations to either vasoconstricting or relaxing agents was investigated in rats maintained in light from 08: 00 to 20: 00 and in darkness from 20: 00 to 08: 00. Rat thoracic aorta rings were obtained from animals sacrificed at four different times of the day. The rat aorta was found to be more sensitive to the constricting effect of phenylephrine at 15: 00, and of 5-hydroxytryptamine at 21: 00. On the other hand, both endothelium-dependent and -independent relaxations were more remarkable at 03: 00 than at other times of the day. These variations represented significant circadian rhythms when analyzed by analysis of variance. Different in vitro responsiveness to these agents might reflect changes in the sensitivity and/or number of related receptors in vascular preparations. In conclusion, the circadian time of animal sacrifice to obtain vascular preparations constitutes an important aspect of the research method and a key determinant of findings. (Chronobiology International, 13(6), 465-475, 1996)     

低氧mtDNA3010A/G基因型变异诱导的lncRNA-mRNA共表达网络变化

目的: 分析mtDNA3010A/G变异在急性缺氧条件下的长链非编码RNA(lncRNA)和信使RNA(mRNA)的共表达网络变化,探讨关键lncRNA和mRNA在低氧诱导的基因表达调控中的作用。方法: 筛选线粒体DNA(mtDNA)3010-5178-10400的基因型组合A-C-C和G-C-C,以骨肉瘤细胞经溴化乙锭处理后形成的无线粒体细胞(ρ⁰206细胞)为供体,构建mtDNA3010A和mtDNA3010G基因型融合细胞。经1%O₂处理24 h后,采用lncRNA-mRNA共表达芯片检测两种融合细胞的差异表达lncRNA和mRNA,荧光定量聚合酶链式法验证差异显著的mRNA,运用生物信息学方法构建lncRNA-mRNA共表达网络,预测差异lncRNA的靶基因,并对差异显著的mRNA和预测靶基因进行基因本体(GO)和京都基因与基因组大百科全书(KEGG)预测分析。结果: 经1%O₂处理24 h后,与mtDNA3010G融合细胞相比,mtDNA3010A融合细胞表达上调的lncRNA有688个,超过2倍的有21个,表达下调的lncRNA有1098个,超过2倍的有4个;表达上调的mRNA有1151个,超过2倍的有14个,表达下调的mRNA有539个,超过2倍的有3个。结论: mtDNA3010A/G基因型变异在缺氧条件下能够影响lncRNA-mRNA调控网络的变化,差异表达的lncRNA和mRNA可能在低氧诱导的基因表达调控网络中发挥重要作用,有望成为从线粒体角度调控低氧反应的靶点。     

纳米二氧化硅复合寒冷对A549细胞毒性及其炎性因子分泌的影响

目的:本研究旨在探讨纳米二氧化硅(Nano-SiO2)颗粒和寒冷复合对人肺腺癌上皮细胞A549细胞毒性及炎性因子分泌的影响。方法:本研究以A549细胞为实验对象,分别用10,50,100,200μg/ml Nano-SiO2颗粒对A549细胞染毒,以及分别在35℃,33℃,31℃条件下对A549细胞进行低温暴露,培养48 h后,观察细胞形态及测定细胞相对存活率。根据单因素分析结果,选出对A549细胞相对存活率有显著降低作用的Nano-SiO2剂量和温度的基础上,按照2×2析因设计实验,分为4组:①37℃对照组;②Nano-SiO2染毒组;③低温暴露组;④Nano-SiO2和低温复合组,不同条件下暴露48 h后,收集细胞上清液采用比色法检测LDH活性,以及ELISA法测定细胞因子白介素-6(IL-6)和白介素-8(IL-8)的水平,采用q RT-PCR法检测细胞IL-6和IL-8的基因表达水平。结果:100μg/ml Nano-SiO2组和31℃低温组能够显著降低A549细胞活性(P<0.01),在复合条件作用下对A549细胞活性抑制最为显著,且炎性因子IL-6和IL-8及m RN...