Proteomics of Blood-Based Therapeutics

Blood-based therapeutics are cellular or plasma components derived from human blood. Their production requires appropriate selection and treatment of the donor and processing of cells or plasma proteins. In contrast to clearly defined, chemically synthesized drugs, blood-derived therapeutics are highly complex mixtures of plasma proteins or even more complex cells. Pathogen transmission by the product as well as changes in the integrity of blood constituents resulting in loss of function or immune modulation are currently important issues in transfusion medicine. Protein modifications can occur during various steps of the production process, such as acquisition, enrichment of separate components (e.g. coagulation factors, cell populations), virus inactivation, conservation, and storage. Contemporary proteomic strategies allow a comprehensive assessment of protein modifications with high coverage, offer capabilities for qualitative and even quantitative analysis, and for high-throughput protein identification. Traditionally, proteomics approaches predominantly relied on two-dimensional gel electrophoresis (2-DE). Even if 2-DE is still state of the art, it has inherent limitations that are mainly based on the physicochemical properties of the proteins analyzed; for example, proteins with extremes in molecular mass and hydrophobicity (most membrane proteins) are difficult to assess by 2-DE. These limitations have fostered the development of mass spectrometry centered on non-gel-based separation approaches, which have proven to be highly successful and are thus complementing and even partially replacing 2-DE-based approaches. Although blood constituents have been extensively analyzed by proteomics, this technology has not been widely applied to assess or even improve blood-derived therapeutics, or to monitor the production processes. As proteomic technologies have the capacity to provide comprehensive information about changes occurring during processing and storage of blood products, proteomics can potentially guide improvement of pathogen inactivation procedures and engineering of stem cells, and may also allow a better understanding of factors influencing the immunogenicity of blood-derived therapeutics. An important development in proteomics is the reduction of inter-assay variability. This now allows the screening of samples taken from the same product over time or before and after processing. Optimized preparation procedures and storage conditions will reduce the risk of protein alterations, which in turn may contribute to better recovery, reduced exposure to allogeneic proteins, and increased transfusion safety.     

Where will pathogen inactivation have the greatest impact?

Blood safety has always been a major task in transfusion medicine. A strategy to obtain this aim should include donor education, donor selection, and testing of blood donations. Pathogen inactivation adds another level of safety. In the fractionation industry, pathogen inactivation methods are mandatory. Several countries also use pathogen‐inactivated plasma – from pools or single donors. Concerning the cellular blood components, there is still no method available for red cell concentrates, whereas methods for platelet concentrates are available in some countries and others are in the pipeline for commercialization. The efficiency of the ‘old’ methods to increase blood safety and the costs of the methods seem to be major obstacles for the introduction of the systems. There are also concerns on product quality and loss of volume during the inactivation process. As the importance of pathogen inactivation is largest in countries with blood donors who carry infections it is impossible to protect against, either due to high incidence of the infection or due to shortage of tests, cost will be a major question when pathogen inactivation is considered. Pathogen inactivation of red cell concentrates will also be a necessity. When pathogen inactivation methods are available for all blood components, they will have great impact to protect the patients in countries where a high percentage of the population is infected by agents transmissible through blood transfusion, and in all situations to protect against new pathogens and ‘old’ pathogens that become more virulent. The total risk of contracting infectious diseases through blood transfusion will probably be important when implementation of new methods for pathogen inactivation is considered.     

Inactivation of Zika virus in plasma and derivatives by four different methods

Zika virus (ZIKV) is an emerging arbovirus with increasing prevalence in recent years. To reduce the risk of ZIKV transmission by transfusion, some mitigation strategies were recommended based on pathogen reduction technologies for blood products. In this study, we aimed to study the efficacy of several common pathogen reduction methods in the inactivation of ZIKV. The fresh frozen plasma and derivatives were spiked with a high titer of ZIKV or Sindbis virus (SINV). Viral titers and ZIKV RNA were measured before and after the inactivation treatment by methylene blue (MB), solvent/detergent (S/D), pasteurization, and low pH. The mean ZIKV infectivity titers in plasma and derivatives were 7.08 ± 0.14, 5.17 ± 0.14, 7.08 ± 0.14, and 5.80 ± 0.14 log₁₀TCID₅₀/mL, respectively before MB, S/D, pasteurization, and low pH inactivation. We found no detectable ZIKV RNA after five successive passages of inoculation on host cells, indicating there is no infectivity after inactivation. Similar inactivation results were observed for SINV. In conclusion, we achieved robust ZIKV inactivation through the four inactivation procedures in several blood products. These findings suggest that the pathogen reduction technologies commonly applied in plasma and derivatives have the capacity to mitigate the risk of ZIKV transmission by transfusion.     

The storage lesions: From past to future

Red blood cell (RBC) concentrates are stored in additive solutions at 4 ^oC for up to 42 days, whereas platelets concentrates (PCs) are stored at 22 ^oC with continuous agitation for up to 5 to 7 days, according national regulations, and the use or not of pathogen inactivation procedures. Storage induces cellular lesion and alters either RBC or platelet metabolism, and is associated with protein alterations. Some age-related alterations prove reversible, while other changes are irreversible, notably following protein oxidation. It is likely that any irreversible damage affects the blood component quality and thus the transfusion efficiency. Nevertheless, there still exists a debate surrounding the impact of storage lesions, for both RBCs and PCs. Uncertainty is not completely resolved. Several studies show a tendency for poorer outcomes to occur in patients receiving older blood products; however, no clear significant association has yet been demonstrated. The present short review aims to promote a better understanding of the occurrence of storage lesions, with particular emphasis on biochemical modifications opening discussions of the future advancement of blood transfusion processes. The paper is also an advocacy for the implementation of an independent international organization in charge of planning and controlling clinical studies in transfusion medicine, in order to base transfusion medicine practices both on security principles, but also on clinical evidences.     

Protecting the blood supply from emerging pathogens: The role of pathogen inactivation

As a consequence of the many blood-safety interventions introduced since the mid-1980s, the major causes of transfusion-associated mortality have shifted from being mainly due to transfusion-transmitted infections (TTIs) to being mainly due to non-infectious serious events such as TRALI, hemolytic reactions, transfusion overload, and graft versus host disease. Thus, TTIs now account for only 10 to 15% of all transfusion associated mortalities! Relevantly, manufacturers of purified plasma protein fractions have, over the same time period, shown that pathogen inactivation technologies can be successfully implemented resulting in little or no transmission of HIV, HCV or HBV since the late 1980s. These technologies, however, cannot be applied to cellular blood components. Thus, new technologies have evolved over the past decade to treat cellular blood components as well as plasma. These technologies, particularly those involving plasma and platelets, have begun to be used in Europe and this proactive paradigm has evolved to become a potential pre-emptive approach for ridding the blood supply of most TTIs (virus, bacteria, and protozoa). However, in order for pathogen inactivation technologies to become widely accepted, they must be shown to be both cost effective and not associated with new risks to recipients!     

Quality management of blood collection

The objective of the quality management in the blood transfusion service is the continuous improvement of the quality of the processes and the products related to the blood donation of the blood donors, the production of the blood components and the transfusion of the blood products to the recipients. The efficacy, the safety and the efficiency of the blood transfusion processes requires that the personnel is educated and trained, that processes are described in standard operating procedures and that these procedures are followed up. When the concept of quality and the improvement of quality is in the mind of all working in the field of blood transfusion medicine, the quality of the blood transfusion service will go upwards, the donors will feel themselves respectfully treated and rewarded, the blood products will become more efficacious and safe, and the patients will receive the best products available. Quality management of blood collection implies the processes on donor recruitment, donor handling and donor vigilance, and the process of donation and screening. For all processes in blood collection, quality indicators should be defined and these data should be monitored, collated, analysed, reported and used in order to improve the quality of the processes.     

Transfusion medicine: Overtime paradigm changes and emerging paradoxes

This essay aims to discuss some aspects of blood transfusion in the perspective of the changes that occurred over time as well as modifications of the paradigms that transformed the activities and the organization of blood transfusion services. Without specific knowledge, pioneers envisioned precision and personalized medicine, rendering transfusion medicine operational. Transfusion medicine is like The Picture of Dorian Grey: always young despite being old and sometimes appearing old-fashioned. Over the years, the transfusion medicine discipline has evolved, and major progress has been achieved, despite some troublesome periods (for example, the tainted blood scandal, and—at the time being—the offending plasma market and the selling of human parts). Transfusion medicine has at all times implemented the rapidly developing biomedical technologies to secure blood components. The safety of blood components has now reached an exceptional level in economically wealthy countries, especially compared to other health care disciplines. Strengthening of the safety has mandated that blood donors and recipients are unrelated, an issue which has eased preservation and fractionation practices; blood is no longer arm-to-arm transfused and neither is whole blood, the commonest component. However, it is interesting to note that a revival is occurring as whole blood is back on stage for certain specific indications, which is one among the many paradoxes encountered while studying this discipline.     

Changing educational paradigms in transfusion medicine

Over the past decades, the fields of activity and knowledge in transfusion medicine have evolved into an array of diverse areas and sub-specialities including immunohaematology, blood component production, haemapheresis, pathogen detection, methods of cell and tissue collection and manipulation, cell conservation and banking, transplant immunology and haemostaseology. Physicians in most clinical disciplines require basic or more advanced knowledge in these fields to meet the requirements of modern medicine. Specialist physicians in transfusion medicine are valuable and competent partners for these related disciplines when it comes to safe, effective and tailored haemotherapy. Transfusion medicine is thus an important qualification at the interfaces of analytical laboratory medicine, pharmaceutical production and clinical disciplines such as internal medicine, anaesthesiology or surgery. In the past, blood transmittable diseases like HIV and hepatitis and adverse reactions to blood and cellular products have led to a complex system of regulatory and technical requirements. Good laboratory practice (GLP), good manufacturing practice (GMP), quality management systems and quality control on the pharmaceutical manufacturer’s level are only a few examples of the standards in today’s blood banking. European directives in the field of blood products, stem cell preparations and tissue have harmonized national regulation and led to higher uniform quality standards for biological preparations in a unified Europe, which is the desired outcome, but which also increases the complexity of this field. By contrast, directives 93/16/EEC, 2001/19/ EC, and 2005/36/EC, the directives of the European Parliament and of the Council on the mutual recognition of professional qualifications of European doctors currently in force, do not include transfusion medicine, blood transfusion or immune haematology at all. Other medical specialities, which like our field, are not common to all member states of the European Union, are listed in the above mentioned directives with the minimum length of training and minimal requirements for the qualifications. Bearing in mind the regional particularities of the medical speciality of transfusion medicine – caused by historical developments, rigidified by national legislations and the urgent need for quality standards also on the educatory level – we support a levelled approach in transfusion medicine education. Irrespective of the required day-to-day responsibilities in the blood field, which may range from basic level experience in haemotherapy, over specific knowledge of immunohaematology and clinical haemopathology, as needed for local blood bank management, up to the highest skill level required to direct a complex transfusion service and/or blood bank at an academic medical centre, transparent service quality requires defined minimum educational standards, which could then be adapted to fit specific national requirements. A long-term objective might be to introduce the transfusion medicine specialisation into the above-mentioned EC directives in order to guarantee quality and facilitate mutual recognition of transfusion medicine qualifications throughout Europe.     

Red blood cell proteomics

Since its discovery in the 17th century, the red blood cell, recognized in time as the critical cell component for survival, has been the focus of much attention. Its unique role in gas exchange (oxygen/CO₂ transport) and its distinct characteristics (absence of nucleus; biconcave cell shape) together with an – in essence – unlimited supply lead to extensive targeted biochemical, molecular and structural studies. A quick PubMed query with the word “erythrocyte” results in 198 013 scientific articles of which 162 are red blood cell proteomics studies, indicating that this new technique has been only recently applied to the red blood cell and related fields. Standard and comparative proteomics have been widely used to study different blood components. A growing body of proteomics literature has since developed, which deals with the characterization of red blood cells in health and disease. The possibility offered by proteomics to obtain a global snapshot of the whole red blood cell protein make-up, has provided unique insights to many fields including transfusion medicine, anaemia studies, intra-red blood cell parasite biology and translational research. While the contribution of proteomics is beyond doubt, a full red blood cell understanding will ultimately require, in addition to proteomics, lipidomics, glycomics, interactomics and study of post-translational modifications. In this review we will briefly discuss the methodology and limitations of proteomics, the contribution it made to the understanding of the erythrocyte and the advances in red blood cell-related fields brought about by comparative proteomics.     

骨科疾病中蛋白质组学的应用进展

背景：蛋白质组学是后基因组时代的新研究领域,它以组织或细胞的全部蛋白质结构与功能为研究对象,通过检测蛋白质来分析各种疾病的重要病理生理变化。 目的：就目前蛋白质组学相关技术在骨科各种疾病中的应用与展望作一综述。 方法：以“组织构建/蛋白质组学/骨科学/椎间盘退变/骨质疏松/骨性关节炎/成骨细胞/破骨细胞/综述”为中文检索词,以“proteomics/orthopedics/review/osteoblasts metabolism/osteoclasts metabolism/ intervertebraldisc degeneration/osteoporosis/osteoarthritis/serum related orthopedic diseases”为英文检索词,检索珠江三角洲数字图书馆联盟期刊全文数据库和Pubmed外文数据库2001年1月至2013年9月有关蛋白质组学在骨科各类疾病当中的基础和临床研究的相关文献。纳入有关报告蛋白质组学在椎间盘病变、骨质疏松症及骨性关节炎等方面的应用的文献。排除重复性研究和不典型报道。 结果与结论：蛋白质组学已成功应用于成骨细胞、破骨细胞代谢,椎间盘退变、骨质疏松症、骨性关节炎及骨科相关疾病的血清等方面,以上所提及的疾病均为各类人群的常见多发病,这些相关的基础临床研究无疑为人类健康带来了巨大的福音,相信蛋白质组学相关技术在基础医学和临床医学中会取得更大的突破,将展示广阔的发展前景。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

How to improve transfusion medicine. A treating physician's perspective.

As transfusion medicine becomes more complex, cooperative strategies are gaining increasing importance in relaying information to the treating physician and in incorporating the treating physician into the education and quality control processes. The broad domain of transfusion medicine is illustrated by the variety of disciplines involved in defining the use of products such as fresh frozen plasma and the newly released solvent-detergent-treated plasma, fibrin glue and highly purified fibrin sealant, and leukoreduced and irradiated blood products. Cooperative efforts among physicians and other personnel of multiple disciplines are essential to ensure appropriate use and continuous evaluation of blood products.     

Détection du génome viral du parvovirus B19 dans les pools de plasmas servant à la préparation du plasma frais congelé traité pour viro-atténuation par solvant–détergent : expérience de l’Afssaps et de l’EFS Aquitaine-Limousin

Since 1998, the Aquitaine-Limousin branch of the French Blood Institute has set up a parvovirus B19 (PV B19) systematic screening on each unit of plasma to be treated by solvent–detergent procedure for virus inactivation. Parvovirus B19 nucleic acid systematic testing in plasma pools became mandatory since 2005 (European monograph “Human plasma” – pooled and treated for virus inactivation). The French competent state authority (AFSSAPS) has decided to introduce this test as a part of the external quality control of labile blood products. This process is related to the harmonization of quality control practice realised on blood products in Europe even if the human plasma pooled and treated for virus inactivation by solvent–detergent is considered in France as a blood labile component. Implementation of this test required a validation step and a close cooperation between AFSSAPS and Aquitaine-Limousin blood transfusion centre. Validation consisted in perfecting a semi-quantitative, real-time nucleic acid testing method with automated extraction. This collaborative study leads us to control 1642 plasma pools. All the results were under the threshold of 10,0 IU/μL. AFSSAPS's results were in agreement with those of Aquitaine-Limousin's blood transfusion center who carry out the parvovirus B19 screening both on fresh frozen plasma units composing the pool and on plasma pools.     

Inactivation des pathogènes des produits sanguins labiles : entre enjeux financiers et conséquences possibles à long terme

Pathogen inactivation of blood products represents a global and major paradigm shift in transfusion medicine. In the next near future, it is likely that most blood products will be inactivated by various physicochemical approaches. The concept of blood safety will be challenged as well as transfusion medicine practice, notably for donor selection or biological qualification. In this context, it seems mandatory to develop analytical economic approaches by assessing costs-benefits ratio of blood transfusion as well as to set up cohorts of patients based on hemovigilance networks allowing rigorous scientific analysis of the benefits and the risks of blood transfusion at short- and long-term.     

Redox proteomics in study of kidney-associated hypertension: new insights to old diseases

Abstract Significance: The kidney helps to maintain low blood pressure in the human body, and impaired kidney function is a common attribute of aging that is often associated with high blood pressure (hypertension). Kidney-related pathologies are important contributors (either directly or indirectly) to overall human mortality. In comparison with other organs, kidney has an unusually wide range of oxidative status, ranging from the well-perfused cortex to near-anoxic medulla. Recent Advances: Oxidative stress has been implicated in many kidney pathologies, especially chronic kidney disease, and there is considerable research interest in oxidative stress biomarkers for earlier prediction of disease onset. Proteomics approaches have been taken to study of human kidney tissue, serum/plasma, urine, and animal models of hypertension. Critical Issues: Redox proteomics, in which oxidative post-translational modifications can be identified in protein targets of oxidative or nitrosative stress, has not been very extensively pursued in this set of pathologies. Future Directions: Proteomics studies of kidney and related tissues have relevance to chronic kidney disease, and redox proteomics, in particular, represents an under-exploited toolkit for identification of novel biomarkers in this commonly occurring pathology. Antioxid. Redox Signal. 17, 1560-1570.     

Plasma fractionation

The first plasma fractionation process was developed in the 1940s by Cohn and co-workers to prepare albumin and immunoglobulins. It relies on sequential precipitation steps at negative temperatures, different ethanol concentrations and various pH to segregate bulk plasma proteins. The ‘Cohn procedure’ has been modified over the years but largely remains the core fractionation process in use up to now. The introduction of cryoprecipitation (thawing of plasma at 2–4°C), as the first plasma fractionation step, to isolate a crude factor-VIII fraction, and the integration of chromatographic steps to extract labile plasma or trace plasma proteins, have eventually shaped the modern fractionation technologies into complex procedures. Viral inactivation and removal treatments are performed to prevent the risks of transmission of enveloped and non-enveloped viruses. Viral inactivation steps are applied most often on pre-purified fractions, the downstream steps being used to remove protein contaminants and viral inactivation agents. Depending upon products and protein stability, treatments include incubations with combinations of solvent (TnBP) and detergent (e.g. Tween 80 or Triton X-100) (S/D), pasteurization (heat-treatment in the liquid state at 60°C for 10 h in the presence of stabilizers), as well as low pH or caprylic acid treatments (these last two treatments are restricted to immunoglobulins). Nanofiltration, to capture viruses on nm-membranes, is typically performed on the final purified protein fractions prior to sterile filtration and aseptic filling. Terminal dry-heat treatment at 80–100°C is used for some coagulation factor preparations. Modern plasma protein products have an unprecedented level of quality and safety when there is careful process control and monitoring together with sensitive test methods to detect potentially harmful contaminants, such as procoagulant impurities. Since the implementation of validated robust viral reduction treatments, plasma products have a high viral safety profile including against emerging agents (WNV, Dengue virus, Chikungunya virus). Experimental spiking studies suggest that several fractionation steps are capable of removing prions causing variant Creutzfeldt-Jakob disease. Alternative fractionation processes based exclusively on chromatography have been developed at pilot scale and need further validations to demonstrate the quality, safety and consistency of resulting plasma products. A mini-pool viral inactivation and fractionation process relying on single-use equipment is also being developed to facilitate the access to safer plasma components, including plasma for transfusion, cryoprecipitate, prothrombin complex, immunoglobulins and fibrin sealant, in particular in developing countries.     

Appropriate blood component usage

Blood transfusion (which includes FFP, platelets, cryoprecipitate and any other blood-derived product) remains an important modality of treatment across all clinical disciplines. A blood transfusion is deemed appropriate when used in an evidence-based fashion and where the effects of the transfusion are felt to outweigh any potential risks and complications that may arise from the transfusion. In certain cases, it may be the best treatment option available, for example plasma exchange in thrombotic thrombocytopenic purpura. However, blood transfusion can result in acute or delayed complications, as well as the risk of transmission of infectious agents. The inappropriate use of blood and blood products increases the risk of transfusion-related complications and adverse events to recipients. It also contributes to shortages of blood products and the possibility of it not being available when required for other patients in an appropriate setting. It is therefore necessary to reduce the unnecessary transfusions through the appropriate clinical use of blood, avoiding unnecessary transfusions, and use of alternatives to transfusion.     

Microbial safety of cellular therapeutics—lessons from over ten years’ experience in microbial safety of platelet concentrates

Bacterial safety of cellular preparations, including blood products and cellular therapeutics, presents ongoing challenges for physicians, manufacturers and regulators. Although there have been many new approaches to enhance the microbial safety of cellular products during the last decade, established methods for microbiological control still need to be fully adapted to the special circumstances of cellular preparations. The experience from transfusion medicine regarding microbial safety of blood components has demonstrated the variety of problems and risk factors for the development of new strategies for microbial safety. Special attention has been given to the prevention and detection of bacterial contamination of platelet concentrates. But so far, none of the targeted strategies for rapid detection or pathogen reduction have become routinely implemented worldwide, in part at least because development and requirements of new technologies and their implementation into the routine setting are a whole different problem. But factors including the short shelf life and nontraditional lot sizes for cellular and gene therapy products are driving the need for rapid microbiological methods. In conclusion, lessons from the microbial safety of platelet concentrates enable us to understand that the detection or reduction in bacteria represents a more difficult challenge in comparison with viruses. Recent regulatory changes demonstrate that we are getting closer to the goal of a shift from the traditional view of sterility evaluation (identify and inactivate anything and everything) to a new thinking about microbiological control.     

Transfusion medicine: debate, field of application, the stakes]

Transfusion medicine has aroused much controversy as to the definition of its field of application, as the Etablissement Fran?ais du Sang (EFS) is being set up. One argument put forward by supporters of transfusion medicine in hospital--including nominal attribution and clinical immunohematology--is the possible influence of blood component producers on the decisions of prescribers. The EFS considers transfusion medicine as the rationale underlying a therapeutic chain applied to blood products and possibly to cell therapy products, within a coherent structure. Any disruption in the chain would result in reduced visibility at each stage of the chain, diluted responsibilities, and less efficient communication between blood collection and actual needs. It is, however, extremely important to clearly distinguish between the activities related to the production of biological products for therapeutic use from the activities of evaluation, in the setting of clinical protocols, which are the clinicians' responsibility. The EFS views the production of blood components as one of the various aspects of its missions as a transfusion medicine operator. The involvement of blood transfusion centers in the development of biotechnologies is a historical reality which justifies EFS's ambition to integrate cell therapy activities within its field of competence. The EFS, with its 18 blood transfusion centers, will have to play a part in health care, research and teaching networks, at the regional or inter-regional level. Irrespective of the progress achieved in substitution products and the evolution to be expected in transfusion-related jobs, transfusion as a discipline must maintain its coherence to be able to better assume its responsibilities.     

Redox proteomics

Abstract Proteins are major targets of reactive oxygen and nitrogen species (ROS/RNS) and numerous post-translational, reversible or irreversible modifications have been characterized, which may lead to a change in the structure and/or function of the oxidized protein. Redox proteomics is an increasingly emerging branch of proteomics aimed at identifying and quantifying redox-based changes within the proteome both in redox signaling and under oxidative stress conditions. Correlation between protein oxidation and human disease is widely accepted, although elucidating cause and effect remains a challenge. Increasing biomedical data have provided compelling evidences for the involvement of perturbations in redox homeostasis in a large number of pathophysiological conditions and aging. Research toward a better understanding of the molecular mechanisms of a disease together with identification of specific targets of oxidative damage is urgently required. This is the power and potential of redox proteomics. In the last few years, combined proteomics, mass spectrometry (MS), and affinity chemistry-based methodologies have contributed in a significant way to provide a better understanding of protein oxidative modifications occurring in various biological specimens under different physiological and pathological conditions. Hence, this Forum on Redox Proteomics is timely. Original and review articles are presented on various subjects ranging from redox proteomics studies of oxidatively modified brain proteins in Alzheimer disease and animal models of Alzheimer and Parkinson disease, to potential new biomarker discovery paradigm for human disease, to chronic kidney disease, to protein nitration in aging and age-related neurodegenerative disorders, electrophile-responsive proteomes of special relevance to diseases involving mitochondrial alterations, to cardiovascular physiology and pathology. Antioxid. Redox Signal. 17, 1487-1489.