生物技术药物的研究开发与产业化现状及前景

本综述了近年来国内外生物技术药物的研究、开发和产业化现状,讨论了我国医药生物技术产业存在的主要问题。提出中国医药生物技术产业发展应遵循自主开发与技术引进相结合及政府引导与市场机制相结合的原则,政府、企业和科技界应发挥各自不同的作用;展望了我国生物药物产业的发展方向和生物技术药物的市场前景。     

X-ray sterilization of biopharmaceutical manufacturing equipment—Extractables profile of a film material and copolyester Tritan™ compared to gamma irradiation

The biopharmaceutical industry gains enormous flexibility in production processes by using sterilized preassembled single-use devices. Gamma irradiation is an established sterilization technology that may be restricted in the future by the availability of ⁶⁰Co as irradiation source and irradiation capacities. X-ray technology is considered an alternative type of radiation for sterilizing SU equipment. In the context of extractables and leachables—one concern connected with the use of single-use process equipment—the effect of X-ray irradiation on the extractables profile of the materials needs to be compared to established gamma irradiation to qualify this alternative technology. An approach is presented to obtain robust and comprehensive extractables data for materials used in SU devices after sterilization either using X-ray or gamma irradiation. A careful selection of the test items and the test design allows a one-to-one comparison of data obtained from a combination of orthogonal analytical techniques. The extractables of a modern SU film material and the copolyester Tritan™ are evaluated. The data presented allow a risk evaluation on the safety of this new sterilization modality for biopharmaceutical applications. It is demonstrated that the extractables profile of a polymer is not affected by the type of irradiation used for sterilization.     

Multivariate PAT solutions for biopharmaceutical cultivation: current progress and limitations

Increasingly elaborate and voluminous datasets are generated by the (bio)pharmaceutical industry and are a major challenge for application of PAT and QbD principles. Multivariate data analysis (MVDA) is required to delineate relevant process information from large multi-factorial and multi-collinear datasets. Here the key role of MVDA for industrial (bio)process data is discussed, with a focus on progress and limitations of MVDA as a PAT solution for biopharmaceutical cultivation processes. MVDA based models were proven useful and should be routinely implemented for bioprocesses. It is concluded that although the highest level of PAT with process control within its design space in real-time during manufacturing is not reached yet, MVDA will be central to reach this ultimate objective for cell cultivations.     

无细胞蛋白表达体系研究进展及在生物制药领域中的应用

作为一种快速高效的体外蛋白合成手段,无细胞蛋白表达体系(Cell-free Protein Synthesis,CFPS)一直以来就被广泛应用于基础生物学领域的研究。与传统的基于细胞的体内表达体系相比,CFPS突破了细胞的生理限制,其可调控性强、对毒性蛋白的耐受力高,使得许多很难在体内合成的复杂蛋白在体外顺利表达。近年来随着研究人员不断对CFPS进行优化,通过简化制备工艺、开发价格低廉的能量再生系统、稳定底物供应、促进蛋白正确折叠等方式,成功研发出生产效率高、成本低廉、反应体积大的表达体系。凭借其高通量和大规模的蛋白表达优势,CFPS为解决生物制药领域中面临的难题提供了新的解决思路,并成功地应用于高通量药物筛选、大规模生产重组蛋白药物、个体化定制肿瘤疫苗等领域,显示出其在生物制药领域的重要应用潜力。     

China's growing biomedical industry

The biomedical industry in China is developing rapidly, and new biological drugs are increasing their share of the pharmaceutical market based on people's needs. China is the largest producer and user of vaccines in the world, but the existing production of vaccines is far from enough to meet the needs of the market. The entire market of biological drugs in China is still smaller than that for traditional medicines and chemicals. Therefore, the biopharmaceutical industry has the potential to be the rising star in the pharmaceutical market in the future.     

Sensitivity of a PER.C6® cell line to bis(2,4-di-tert-butylphenyl)phosphate and evaluation of a new,biocompatible single-use film

Single-use technologies have brought numerous advantages to the biopharmaceutical industry. In particular, single-use bags made from multi-layered polymeric films have been adopted for cell culture and liquid handling operations in place of traditional stainless-steel systems. Despite the advantages, leachable compounds originating from the film's materials of construction present a new challenge. In 2013, bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) was identified as a common leachable from several single-use bags that is detrimental to the growth and viability of many Chinese hamster ovary (CHO) cell lines. While much work has been completed to characterize CHO cell sensitivity to bDtBPP, little has been done to characterize its impact on other important production cell lines, particularly PER.C6®. This publication investigates inconsistent cell growth observed in a PER.C6® cell line during bioprocess development. The growth inhibition was linked to leachable migration from Bioclear™ 10, a single-use film from Cytiva (formerly GE Healthcare) that was used for cell expansion. It was shown that the PER.C6® cells displayed a sensitivity to bDtBPP, comparable to that observed in sensitive CHO cell lines. Finally, biocompatibility of PER.C6® with Cytiva's new Fortem film was evaluated, demonstrating that Fortem™ film is a suitable single-use technology for culturing PER.C6® cells.     

Ectopic expression of human mTOR increases viability, robustness, cell size, proliferation, and antibody production of chinese hamster ovary cells

Engineering of mammalian production cell lines to improve titer and quality of biopharmaceuticals is a top priority of the biopharmaceutical manufacturing industry providing protein therapeutics to patients worldwide. While many engineering strategies have been successful in the past decade they were often based on the over‐expression of a single transgene and therefore limited to addressing a single bottleneck in the cell's production capacity. We provide evidence that ectopic expression of the global metabolic sensor and processing protein mammalian target of rapamycin (mTOR), simultaneously improves key bioprocess‐relevant characteristics of Chinese hamster ovary (CHO) cell‐derived production cell lines such as cell growth (increased cell size and protein content), proliferation (increased cell‐cycle progression), viability (decreased apoptosis), robustness (decreased sensitivity to sub‐optimal growth factor and oxygen supplies) and specific productivity of secreted human glycoproteins. Cultivation of mTOR‐transgenic CHO‐derived cell lines engineered for secretion of a therapeutic IgG resulted in antibody titers of up to 50 pg/cell/day, which represents a four‐fold increase compared to the parental production cell line. mTOR‐based engineering of mammalian production cell lines may therefore have a promising future in biopharmaceutical manufacturing of human therapeutic proteins. Biotechnol. Bioeng. 2011; 108:853–866. © 2010 Wiley Periodicals, Inc.     

Multivariate statistical monitoring as applied to clean‐in‐place (CIP) and steam‐in‐place (SIP) operations in biopharmaceutical manufacturing

Multivariate statistical process monitoring (MSPM) is becoming increasingly utilized to further enhance process monitoring in the biopharmaceutical industry. MSPM can play a critical role when there are many measurements and these measurements are highly correlated, as is typical for many biopharmaceutical operations. Specifically, for processes such as cleaning‐in‐place (CIP) and steaming‐in‐place (SIP, also known as sterilization‐in‐place), control systems typically oversee the execution of the cycles, and verification of the outcome is based on offline assays. These offline assays add to delays and corrective actions may require additional setup times. Moreover, this conventional approach does not take interactive effects of process variables into account and cycle optimization opportunities as well as salient trends in the process may be missed. Therefore, more proactive and holistic online continued verification approaches are desirable. This article demonstrates the application of real‐time MSPM to processes such as CIP and SIP with industrial examples. The proposed approach has significant potential for facilitating enhanced continuous verification, improved process understanding, abnormal situation detection, and predictive monitoring, as applied to CIP and SIP operations. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:505–515, 2014     

Accelerated CMC workflows to enable speed to clinic in the COVID-19 era: A multi-company view from the biopharmaceutical industry

The COVID-19 pandemic has placed unprecedented pressure on biopharmaceutical companies to develop efficacious preventative and therapeutic treatments, which is unlikely to abate in the coming years. The importance of fast progress to clinical evaluation for treatments, which tackle unmet medical needs puts strain on traditional product development timelines, which can take years from start to finish. Although previous work has been successful in reducing phase 1 timelines for recombinant antibodies, through utilization of the latest technological advances and acceptance of greater business risk or costs, substantially faster development is likely achievable without increased risk to patients during initial clinical evaluation. To optimize lessons learned from the pandemic and maximize multi-stakeholder (i.e., patients, clinicians, companies, regulatory agencies) benefit, we conducted an industry wide benchmarking survey in September/October 2021. The aims of this survey were to: (i) benchmark current technical practices of key process and product development activities related to manufacturing of therapeutic proteins, (ii) understand the impact of changes implemented in COVID-19 accelerated Ab programs, and whether any such changes can be retained as part of sustainable long-term business practices and (iii) understand whether any accelerative action(s) taken have (negatively) impacted the wider development process. This article provides an in-depth analysis of this data, ultimately highlighting an industry perspective of how biopharmaceutical companies can sustainably adopt new approaches to therapeutic protein development and production.     

Endotoxin inactivation via steam‐heat treatment in dilute simethicone emulsions used in biopharmaceutical processes

Simethicone emulsion is used to regulate foaming in cell culture operations in biopharmaceutical processes. It is also a potential source of endotoxin contamination. The inactivation of endotoxins in dilute simethicone emulsions was assessed as a function of time at different steam temperatures using a Limulus amebocyte lysate kinetic chromogenic technique. Endotoxin inactivation from steam‐heat treatment was fit to a four‐parameter double exponential decay model, which indicated that endotoxin inactivation was biphasic, consisting of fast and slow regimes. In the fast regime, temperature‐related effects were dominant. Transitioning into the slow regime, the observed temperature dependence diminished, and concentration‐related effects became increasingly significant. The change in the Gibbs free energy moving through the transition state indicated that a large energy barrier must be overcome for endotoxin inactivation to occur. The corresponding Arrhenius pre‐exponential factor was >>10¹² s^?1 suggesting that endotoxins in aqueous solution exist as aggregates. The disorder associated with the endotoxin inactivation reaction pathway was assessed via the change in entropy moving through the transition state. This quantity was positive indicating that endotoxin inactivation may result from hydrolysis of individual endotoxin molecules, which perturbs the conformation of endotoxin aggregates, thereby modulating the biological activity observed. Steam‐heat treatment decreased endotoxin levels by 1–2 logarithm (log) reduction (LRV), which may be practically relevant depending on incoming raw material endotoxin levels. Antifoam efficiency and cell culture performance were negligibly impacted following steam‐heat treatment. The results from this study show that steam‐heat treatment is a viable endotoxin control strategy that can be implemented to support large‐scale biopharmaceutical manufacturing. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1145–1160, 2014     

英国生物技术产业创新创业生态系统的构建与启示

生物技术产业是各大经济体在21世纪优先发展的战略性产业,逐步成为世界经济的主导产业.英国生物技术产业规模在欧洲排名第一,创新创业文化浓厚.通过孵化器运营案例详细阐述英国生物技术产业发展的生态系统构建.针对我国生物技术产业创新环境提出相应建议,以期对我国生物医药产业的创新创业发展提供借鉴.     

Bioprocess engineering: now and beyond 2000

Abstract: Bioprocess engineering may be defined as the translation of life-science discoveries into practical products, processes, or systems capable of serving the needs of society. It is a critical link from discovery to commercialization. Current bioprocess engineering is primarily focused on biopharmaceutical products of high dollar value per gram such as erythropoietin or growth hormones. However, other products of current interest include ethanol, amino acids, organic acids, antibiotics, and specialty chemicals. Current challenges for increased use of bioprocesses for producing bulk and semi-bulk chemicals include both technical and infrastructural barriers. Technical barriers are easy to identify and at times can be overcome by engineering improvements or changes brought about radical developments in science (e.g. recombinant DNA). Infrastructural barriers, such as raw-material substitutions or educational limitations are more difficult to define and change. Recently the National Academy of Sciences examined barriers to bioprocess engineering and issued a report entitled: "Putting Biotechnology to Work: Bioprocess Engineering". A key recommendation was the establishment of a coordinated long-range plan of research, development, training and education in bioprocess engineering involving participation by industry, academe and the federal government. The report was the first national analysis devoted entirely to bioprocess engineering and covered new topics such as space bioprocess engineering. Other topics covered by the author include the current state of the US chemical industry and future directions in three promising areas of bioprocess engineering environmental bioprocess engineering, marine bioprocess engineering and microsystem bioprocess engineering.     

The birth pangs of monoclonal antibody therapeutics: The failure and legacy of Centoxin

This paper examines the development and termination of nebacumab (Centoxin®), a human IgM monoclonal antibody (mAb) drug frequently cited as one of the notable failures of the early biopharmaceutical industry. The non-approval of Centoxin in the United States in 1992 generated major concerns at the time about the future viability of any mAb therapeutics. For Centocor, the biotechnology company that developed Centoxin, the drug posed formidable challenges in terms of safety, clinical efficacy, patient selection, the overall economic costs of health care, as well as financial backing. Indeed, Centocor''s development of the drug brought it to the brink of bankruptcy. This article shows how many of the experiences learned with Centoxin paved the way for the current successes in therapeutic mAb development.     

Patent pools and clearinghouses in the life sciences

The biopharmaceutical industry is slowly absorbing the idea of collaborative patent licensing models. Recently, two patent pools for developing countries have been launched: the Pool for Open Innovation against Neglected Tropical Diseases initiated by GlaxoSmithKline (GSK), which is referred to as the BIO Ventures for Global Health (BVGH) pool, and the Medicines Patent Pool (MPP) initiated by UNITAID. Various organizations have recommended using pools or clearinghouses beyond the humanitarian dimension where many patents are owned by many different actors. As a first attempt, MPEG LA, which administers patent pools in various technology fields, is now setting up a clearinghouse for patents related to molecular diagnostics. These examples as well as the results from an empirical study provide useful insights for the design and administration of future pools and clearinghouses in the life sciences.     

The birth pangs of monoclonal antibody therapeutics

This paper examines the development and termination of nebacumab (Centoxin®), a human IgM monoclonal antibody (mAb) drug frequently cited as one of the notable failures of the early biopharmaceutical industry. The non-approval of Centoxin in the United States in 1992 generated major concerns at the time about the future viability of any mAb therapeutics. For Centocor, the biotechnology company that developed Centoxin, the drug posed formidable challenges in terms of safety, clinical efficacy, patient selection, the overall economic costs of health care, as well as financial backing. Indeed, Centocor's development of the drug brought it to the brink of bankruptcy. This article shows how many of the experiences learned with Centoxin paved the way for the current successes in therapeutic mAb development.     

The production of biopharmaceuticals in plant systems

Biopharmaceuticals present the fastest growing segment in the pharmaceutical industry, with an ever widening scope of applications. Whole plants as well as contained plant cell culture systems are being explored for their potential as cheap, safe, and scalable production hosts. The first plant-derived biopharmaceuticals have now reached the clinic. Many biopharmaceuticals are glycoproteins; as the Golgi N-glycosylation machinery of plants differs from the mammalian machinery, the N-glycoforms introduced on plant-produced proteins need to be taken into consideration. Potent systems have been developed to change the plant N-glycoforms to a desired or even superior form compared to the native mammalian N-glycoforms. This review describes the current status of biopharmaceutical production in plants for industrial applications. The recent advances and tools which have been utilized to generate glycoengineered plants are also summarized and compared with the relevant mammalian systems whenever applicable.     

Polyclonal antibodies for specific detection of tobacco host cell proteins can be efficiently generated following RuBisCO depletion and the removal of endotoxins

The production of biopharmaceutical proteins in plants requires efficient downstream processing steps that remove impurities such as host cell proteins (HCPs) and adventitious endotoxins produced by bacteria during transient expression. We therefore strived to develop effective routines for endotoxin removal from plant extracts and the subsequent use of the extracts to generate antibodies detecting a broad set of HCPs. At first, we depleted the superabundant protein ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) for which PEG precipitation achieved the best results, preventing a dominant immune reaction against this protein. We found that a mixture of sera from rabbits immunized with pre‐depleted or post‐depleted extracts detected more HCPs than the individual sera used alone. We also developed a powerful endotoxin removal procedure using Polymyxin B for extracts from wild type plants or a combination of fiber‐flow filtration and EndoTrap Blue for tobacco plants infiltrated with Agrobacterium tumefaciens. The antibodies we generated will be useful for quality and performance assessment in future process development and the methods we present can easily be transferred to other expression systems rendering them useful in the field of plant molecular farming.     

Is 2020 the year when primatologists should cancel fieldwork?

Year 2020 has brought the greatest global pandemic to hit the world since the end of the First World War. The severe acute respiratory syndrome coronavirus 2 and the resulting disease named coronavirus disease 2019 has brought the world to its knees both financially and medically. The American Society of Primatologists has postponed their annual meetings from the end of May 2020 until the end of September 2020, while the International Primatological Society have postponed their biennial congress from August 2020 to August 2021, which has also resulted in their 2022 meetings in Malaysia being pushed back until 2023. Here, I explore the potential dangers of pursuing any primate fieldwork during this pandemic on our study species, their ecosystems, and local peoples. I believe that the risk of bringing this virus into our study ecosystems is too great and that primatologists should cancel all field research until the pandemic ends or a vaccine/reliable treatment is widely available. This is the year we all must become One Health practitioners!     

Utilizing targeted integration CHO pools to potentially accelerate the GMP manufacturing of monoclonal and bispecific antibodies

Monoclonal antibodies (mAbs) are effective therapeutic agents against many acute infectious diseases including COVID-19, Ebola, RSV, Clostridium difficile, and Anthrax. mAbs can therefore help combat a future pandemic. Unfortunately, mAb development typically takes years, limiting its potential to save lives during a pandemic. Therefore “pandemic mAb” timelines need to be shortened. One acceleration tool is “deferred cloning” and leverages new Chinese hamster ovary (CHO) technology based on targeted gene integration (TI). CHO pools, instead of CHO clones, can be used for Phase I/II clinical material production. A final CHO clone (producing the mAb with a similar product quality profile and preferably with a higher titer) can then be used for Phase III trials and commercial manufacturing. This substitution reduces timelines by ~3 months. We evaluated our novel CHO TI platform to enable deferred cloning. We created four unique CHO pools expressing three unique mAbs (mAb1, mAb2, and mAb3), and a bispecific mAb (BsAb1). We then performed single-cell cloning for mAb1 and mAb2, identifying three high-expressing clones from each pool. CHO pools and clones were inoculated side-by-side in ambr15 bioreactors. CHO pools yielded mAb titers as high as 10.4 g/L (mAb3) and 7.1 g/L (BsAb1). Subcloning yielded CHO clones expressing higher titers relative to the CHO pools while yielding similar product quality profiles. Finally, we showed that CHO TI pools were stable by performing a 3-month cell aging study. In summary, our CHO TI platform can increase the speed to clinic for a future “pandemic mAb.”     

Bioprocessing in the Digital Age: The Role of Process Models

In this age of technology, the vision of manufacturing industries built of smart factories is not a farfetched future. As a prerequisite for Industry 4.0, industrial sectors are moving towards digitalization and automation. Despite its tremendous growth reaching a sales value of worth $188 billion in 2017, the biopharmaceutical sector distinctly lags in this transition. Currently, the challenges are innovative market disruptions such as personalized medicine as well as increasing commercial pressure for faster and cheaper product manufacturing. Improvements in digitalization and data analytics have been identified as key strategic activities for the next years to face these challenges. Alongside, there is an emphasis by the regulatory authorities on the use of advanced technologies, proclaimed through initiatives such as Quality by Design (QbD) and Process Analytical Technology (PAT). In the manufacturing sector, the biopharmaceutical domain features some of the most complex and least understood processes. Thereby, process models that can transform process data into more valuable information, guide decision‐making, and support the creation of digital and automated technologies are key enablers. This review summarizes the current state of model‐based methods in different bioprocess related applications and presents the corresponding future vision for the biopharmaceutical industry to achieve the goals of Industry 4.0 while meeting the regulatory requirements.