A 3-year experience of quality control and quality assurance in the multisite delivery of a lymphocyte-based cellular therapy for renal cell carcinoma

Autolymphocyte therapy (ALT) is outpatient-based adoptive immunotherapy using ex vivo-activated memory T-cells. To support the safe and reproducible delivery of ALT at three cell processing facilities (Boston, MA; Atlanta, GA; Orange, CA) we created a comprehensive quality assurance/quality control program compliant with recent FDA guidance relevant to activated lymphocytes and somatic cell therapies. Each facility performed extensive QC testing to ensure sterility, viability, and proper cell yield. Additonally, several QC tests were performed at Cellocr's centralized reference laboratory to monitor cell potency and identity of the ex vivo-processed lymphocytes. We report here the successful implementation of this QA/QC program for ALT which has resulted in the safe preparation and delivery of cell infusion products amounting to over 3600 treatments at seven clinical sites nationwide. We believe this program will serve as a model for other cellular therapies.     

Quality assurance and quality control processes: summary of a metabolomics community questionnaire

Introduction

The Metabolomics Society Data Quality Task Group (DQTG) developed a questionnaire regarding quality assurance (QA) and quality control (QC) to provide baseline information about current QA and QC practices applied in the international metabolomics community.

Objectives

The DQTG has a long-term goal of promoting robust QA and QC in the metabolomics community through increased awareness via communication, outreach and education, and through the promotion of best working practices. An assessment of current QA and QC practices will serve as a foundation for future activities and development of appropriate guidelines.

Method

QA was defined as the set of procedures that are performed in advance of analysis of samples and that are used to improve data quality. QC was defined as the set of activities that a laboratory does during or immediately after analysis that are applied to demonstrate the quality of project data. A questionnaire was developed that included 70 questions covering demographic information, QA approaches and QC approaches and allowed all respondents to answer a subset or all of the questions.

Result

The DQTG questionnaire received 97 individual responses from 84 institutions in all fields of metabolomics covering NMR, LC-MS, GC-MS, and other analytical technologies.

Conclusion

There was a vast range of responses concerning the use of QA and QC approaches that indicated the limited availability of suitable training, lack of Standard Operating Procedures (SOPs) to review and make decisions on quality, and limited use of standard reference materials (SRMs) as QC materials. The DQTG QA/QC questionnaire has for the first time demonstrated that QA and QC usage is not uniform across metabolomics laboratories. Here we present recommendations on how to address the issues concerning QA and QC measurements and reporting in metabolomics.

     

Critical testing and parameters for consideration when manufacturing and evaluating tumor–associated antigen-specific T cells

The past year has seen remarkable translation of cellular and gene therapies, with U.S. Food and Drug Administration (FDA) approval of three chimeric antigen receptor (CAR) T-cell products, multiple gene therapy products, and the initiation of countless other pivotal clinical trials. What makes these new drugs most remarkable is their path to commercialization: they have unique requirements compared with traditional pharmaceutical drugs and require different potency assays, critical quality attributes and parameters, pharmacological and toxicological data, and in vivo efficacy testing. What's more, each biologic requires its own unique set of tests and parameters. Here we describe the unique tests associated with ex vivo–expanded tumor-associated antigen T cells (TAA-T). These tests include functional assays to determine potency, specificity, and identity; tests for pathogenic contaminants, such as bacteria and fungus as well as other contaminants such as Mycoplasma and endotoxin; tests for product characterization, tests to evaluate T-cell persistence and product efficacy; and finally, recommendations for critical quality attributes and parameters associated with the expansion of TAA-Ts.     

Non-viral gene therapy for bone tissue engineering

The possibilities of using gene therapy for bone regeneration have been extensively investigated. Improvements in the design of new transfection agents, combining vectors and delivery/release systems to diminish cytotoxicity and increase transfection efficiencies have led to several successful in vitro, ex vivo and in vivo strategies. These include growth factor or short interfering ribonucleic acid (siRNA) delivery, or even enzyme replacement therapies, and have led to increased osteogenic differentiation and bone formation in vivo. These results provide optimism to consider use in humans with some of these gene-delivery strategies in the near future.     

Assemblage diversity,cell density and within-slide variability: Implications for quality assurance/quality control and uncertainty assessment in diatom-based monitoring

This study was undertaken to evaluate the variability associated with the microscope analysis step in the application of the Eastern Canadian Diatom Index (IDEC: Indice Diatomées de l’Est du Canada), with the general objective of developing a suitable quality assurance/quality control (QA/QC) program for this biological index. For this purpose, we estimated within-slide variability (replicability) and inter-analysts variability (reproducibility), as a function of diatom assemblage diversity and slide cell density. Overall, our results show that variability associated with diatom assemblage characterization is low, which ensures that IDEC scores reflect environmental changes rather than variability at the microscope analysis step. The main recommendations ensuing from this study are (for the IDEC in particular but also for diatom-based monitoring in general):

• (1)An error term of ±2 IDEC units corresponding to the within-slide variability (replicability) should accompany all reported IDEC scores.
• (2)A deviation of ±3 points from the audit's IDEC scores should be considered as an acceptable difference. Considering the above-mentioned estimated error term of ±2 associated with all IDEC scores, an overall deviation of 7 would still be satisfactory.
• (3)Samples showing low diversity (Hill's N2 ≤5) should automatically be submitted for QA/QC.
• (4)A Bray–Curtis (analyst vs audit) similarity of ≥60% should also be included as a QA/QC criterion, and should increase to ≥70% for poorly diversified assemblages (Hill's N2 ≤5).
• (5)A diatom valve density of ≤15 per field of view should be targeted in order to reduce variability at the enumeration step.

The results of this study illustrate how a relatively simple and straightforward approach to QA/QC can greatly strengthen the reliability of ecological inferences from an index based on a group of organisms with a high taxonomical diversity. It also highlights the importance of regular communication between analysts in order to maintain a high degree of concordance within taxonomical identification.     

Human somatic cell gene therapy

The prelude to successful human somatic gene therapy, i.e. the efficient transfer and expression of a variety of human genes into target cells, has already been accomplished in several systems. Safe methods have been devised to do this using non-viral and viral vectors. Potentially therapeutic genes have been transferred into many accessible cell types, including hematopoietic cells, hepatocytes and cancer cells, in several different approaches to ex vivo gene therapy. Successful in vivo gene therapy requires improvements in tissuetargeting and new vector design, which are already being sought. Gene-transfer protocols have been approved for human use in inherited diseases, cancer and acquired disorders. Althouth the results of these trials to date have been somewhat disappointing, human somatic cell gene therapy promises to be an effective addition to the arsenal of approaches to the therapy of many human diseases in the 21st century if not sooner.     

Guidelines and considerations for the use of system suitability and quality control samples in mass spectrometry assays applied in untargeted clinical metabolomic studies

Background

Quality assurance (QA) and quality control (QC) are two quality management processes that are integral to the success of metabolomics including their application for the acquisition of high quality data in any high-throughput analytical chemistry laboratory. QA defines all the planned and systematic activities implemented before samples are collected, to provide confidence that a subsequent analytical process will fulfil predetermined requirements for quality. QC can be defined as the operational techniques and activities used to measure and report these quality requirements after data acquisition.

Aim of review

This tutorial review will guide the reader through the use of system suitability and QC samples, why these samples should be applied and how the quality of data can be reported.

Key scientific concepts of review

System suitability samples are applied to assess the operation and lack of contamination of the analytical platform prior to sample analysis. Isotopically-labelled internal standards are applied to assess system stability for each sample analysed. Pooled QC samples are applied to condition the analytical platform, perform intra-study reproducibility measurements (QC) and to correct mathematically for systematic errors. Standard reference materials and long-term reference QC samples are applied for inter-study and inter-laboratory assessment of data.

     

Isolation and therapeutic potential of human haemopoietic stem cells

The haemopoietic stem cell (HSC) has long been regarded as an archetypal, tissue specific, stem cell, capable of completely regenerating haemopoiesis after myeloablation. It has proved relatively easy to harvest HSC, from bone marrow or peripheral blood. In turn, isolation of these cells has allowed therapeutic stem cell transplantation protocols to be developed, that capitalise on their prodigious self renewal and proliferative capabilities. Ex vivo approaches have been described to isolate, genetically manipulateand expand pluripotent stem cell subsets. These techniques have been crucial to the development of gene therapy, and may allow adults to enjoy the potential advantages of cord blood transplantation. Recently, huge conceptual changes have occurred in stem cell biology. In particular, the dogma that, in adults, stem cells are exclusively tissue restricted has been questioned and there is great excitement surrounding the potential plasticity of these cells, with the profound implications that this has, for developing novel cellular therapies. Mesenchymal stem cells, multipotent adult progenitor cells and embryonic stem cells are potential sources of cells for transplantation purposes. These cells may be directed toproduce HSC, in vitro and in the future may be used for therapeutic, or drug development, purposes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Endothelial progenitor cells: Characterization, <Emphasis Type="Italic">in vitro</Emphasis> expansion,and prospects for autologous cell therapy

Injection of hematopoietic stem cells or endothelial progenitor cells (EPCs) expanded ex vivo has been shown to augment neovascularization in adult patients, but the precise origin and identity of the cell population responsible for these clinical benefits are controversial. The limited quantity of EPCs in the circulation has been the main obstacle to clinical trials. Several authors have therefore attempted to expand these cells ex vivo in order to obtain a homogeneous cell therapy product. One possible means of expanding EPCs ex vivo is to activate the thrombin receptor PAR-1 with the specific peptide SFLLRN. Indeed, PAR-1 activation promotes cell proliferation and C-X-C chemokine receptor type 4 (CXCR4) dependent migration and differentiation, with an overall angiogenic effect. This review summarizes the results and rationale of clinical trials of angiogenic therapy, the nature of EPCs, the different methods of ex vivo expansion, and current methods of quantification.     

The quality control system

Quality Control System: an understanding of analytical error; synthetic QC material; a set of QC rules; a process to follow if the rules signal. Quality Control (QC) Sera: reconstitution - staff trained; stability tested - post reconstitution and frozen. QC Rules: rules documented - basis of adoption; action to follow in case of failure documented; evidence of this procedure being used in place; are QC rules defined for both batch and continuous analysis - how is a 'run' defined for a continuous analytical process; means and standard deviations (SDs) of controls based on sufficient data points and reflects true state of system; evidence of staff training in the interpretation of QC rules; process documented; evidence of training of staff; evidence of regular review of Internal QC results. Patient-based QC Procedures in place: if delta check/anion gap/rerun of samples used, then a documented procedure to describe the process and evidence of it being in use; critical values - documented and evidence of use and documentation. Action on QC Rule Failure: documented process to follow with patient samples if control failure occurs; evidence that procedure has been followed in instances of control failure. External Quality Assessment (EQA) Program: Integration of Internal and External QC data.     

Fromin vitro toin vivo. Progress in the use of cultured cells for human therapy

The use of cultured cells with the ultimate goal of using the cells or their products for human therapy has experienced an exponential growth during the last decade. Stable cell cultures have been established and genetically modified to obtain high quality products for protein replacement therapy or vaccines. Cells have also been directly isolated from the human organism and, after their expansionin vitro, been retransferred as skin grafts for treatment of burns or for cancer therapy by activated lymphocytes. With the explosive development of molecular biology techniques, it is now possible to genetically modifyex vivo, cells derived from the human body. These modifications should allow targeted expression of therapeutic genes into specific cells which will, upon retransfer to the body, exert their therapeutic action in a diseased organism.Abbreviations ADA adenosine deaminase - GM-CSF granulocyte-macrophage colony-stimulating factor - IFN interferon - IL interleukin - TIL tumor infiltrating lymphocytes     

Efficient and persistent transduction of exocrine and endocrine pancreas by adeno-associated virus type 8

Efficient delivery of therapeutic proteins into the pancreas represents a major obstacle to gene therapy of pancreatic disorders. The current study compared the efficiency of recombinant lentivirus and adeno-associated virus (AAV) serotypes 1, 2, 5, 8 vectors delivered by intrapancreatic injection for gene transfer in vivo. Our results indicate that lentivirus and AAV 1, 2, 8 are capable of transducing pancreas with the order of efficiency AAV8 >>AAV1 > AAV2 ≥ lentivirus, whereas AAV5 was ineffective. AAV8 resulted in an efficient, persistent (150 days) and dose-dependent transduction in exocrine acinar cells and endocrine islet cells. Pancreatic ducts and blood vessels were also transduced. Extrapancreatic transduction was restricted to liver. Leukocyte infiltration was not observed in pancreas and blood glucose levels were not altered. Thus, AAV8 represents a safe and effective vehicle for therapeutic gene transfer to pancreas in vivo.     

An audit of a hospital-based Doppler ultrasound quality control protocol using a commercial string Doppler phantom

Results from a four-year audit of a Doppler quality assurance (QA) program using a commercially available Doppler string phantom are presented. The suitability of the phantom was firstly determined and modifications were made to improve the reliability and quality of the measurements. QA of Doppler ultrasound equipment is very important as data obtained from these systems is used in patient management. It was found that if the braided-silk filament of the Doppler phantom was exchanged with an O-ring rubber filament and the velocity range below 50 cm/s was avoided for Doppler quality control (QC) measurements, then the maximum velocity accuracy (MVA) error and intrinsic spectral broadening (ISB) results obtained using this device had a repeatability of 18 ± 3.3% and 19 ± 3.5%, respectively. A consistent overestimation of the MVA of between 12% and 56% was found for each of the tested ultrasound systems. Of more concern was the variation of the overestimation within each respective transducer category: MVA errors of the linear, curvilinear and phased array probes were in the range 12.3–20.8%, 32.3–53.8% and 27–40.7%, respectively. There is a dearth of QA data for Doppler ultrasound; it would be beneficial if a multicentre longitudinal study was carried out using the same Doppler ultrasound test object to evaluate sensitivity to deterioration in performance measurements.     

Quality control issues in point of care testing

* Quality Control (QC) in Point of Care Testing (PoCT) is often thought of as a complex issue; however intelligent system analysis can simplify matters and greatly increase the chances of a well controlled system. What we want to achieve is a QC program which adequately controls the PoCT system, but does not excessively contribute to the operating costs or complexity of maintaining a PoCT instrument, or network of instruments. * Don't neglect effective pre-analytical work: good documentation, operator training, monitoring, and analyser maintenance programs are essential, as for any analyser. * Look closely at your analyser: Is it a "laboratory type" instrument or cartridge or strip based? Can it perform multiple test types or a single test only? How is it calibrated? Does it have built in self-check capabilities or an electronic check cartridge? Is the sample in contact with the instrument? What are the cartridge/strip/reagent storage requirements? * Establish where the analysis is taking place and which system component is involved. * Tailor your QC program to target this component, but still check the system as a whole. * A common approach is to check cartridges/strips on delivery and run a QA sample at least monthly to check storage conditions and operator performance. If there is no independent electronic instrument check, daily QC checks are also recommended. * Don't be afraid to stray beyond conventional QC models if necessary. Some PoCT systems are not adequately controlled by the application of conventional QC alone.     

Gene therapy moves forward - The Second International Meeting on Gene and Cell Therapies of Arthritis and Related Disorders, 17-18 May 2001, Montpellier, France

The field of gene therapy for bone and joint disorders has grown considerably over the last two and a half years. Investigators have shown that ex vivo or in vivo gene transfer is highly effective in blocking arthritis or facilitating repair of damaged cartilage or bone. The feasibility of applying gene therapy for the treatment of arthritis in humans has also been demonstrated. Thus, gene therapy appears poised to make significant contributions to the clinical treatment of joint and bone diseases in the near future.     

Multicenter cell processing for cardiovascular regenerative medicine applications: the Cardiovascular Cell Therapy Research Network (CCTRN) experience

Background aimsMulticenter cellular therapy clinical trials require the establishment and implementation of standardized cell-processing protocols and associated quality control (QC) mechanisms. The aims here were to develop such an infrastructure in support of the Cardiovascular Cell Therapy Research Network (CCTRN) and to report on the results of processing for the first 60 patients.MethodsStandardized cell preparations, consisting of autologous bone marrow (BM) mononuclear cells, prepared using a Sepax device, were manufactured at each of the five processing facilities that supported the clinical treatment centers. Processing staff underwent centralized training that included proficiency evaluation. Quality was subsequently monitored by a central QC program that included product evaluation by the CCTRN biorepositories.ResultsData from the first 60 procedures demonstrated that uniform products, that met all release criteria, could be manufactured at all five sites within 7 h of receipt of BM. Uniformity was facilitated by use of automated systems (the Sepax for processing and the Endosafe device for endotoxin testing), standardized procedures and centralized QC.ConclusionsComplex multicenter cell therapy and regenerative medicine protocols can, where necessary, successfully utilize local processing facilities once an effective infrastructure is in place to provide training and QC.     

Therapeutic bacteriophages as a rescue treatment for drug-resistant infections – an in vivo studies overview

Bacteriophages, highly prevalent in all environments, have found their use in medicine as an alternative or complement to antibiotics. The therapeutic use of bacteriophages was particularly popular in the 1920s and 1930s, until the discovery and introduction of antibiotics. Due to the dynamic growth of antibiotic resistance among bacterial strains, numerous international institutions (such as the FDA) have declared the search for novel treatment modalities to be of the highest priority. To date, bacteriophage therapy has not been registered for general use in Western countries. The regulation of biological medicinal products (within medicinal product regulation) does not contain a specific documentation frame for bacteriophages (only for vaccines, blood derived products, etc.) which, as active substances, need to meet specific requirements. Recently, the FDA allowed bacteriophage therapy to be used in the United States, via the Emergency Investigational New Drug scheme; clinical trials to compare the safety and efficacy of bacteriophage therapy are also permitted. To date, several therapeutic products of this type have made it to phase I or II; some clinical programmes have also been completed. This article cites numerous animal model studies and registered clinical trials, showing the safety and effectiveness of bacteriophage therapy, including infections caused by bacterial strains resistant to antibiotic treatment.     

Broadening the reach and investigating the potential of prime editors through fully viral gene-deleted adenoviral vector delivery

Prime editing is a recent precision genome editing modality whose versatility offers the prospect for a wide range of applications, including the development of targeted genetic therapies. Yet, an outstanding bottleneck for its optimization and use concerns the difficulty in delivering large prime editing complexes into cells. Here, we demonstrate that packaging prime editing constructs in adenoviral capsids overcomes this constrain resulting in robust genome editing in both transformed and non-transformed human cells with up to 90% efficiencies. Using this cell cycle-independent delivery platform, we found a direct correlation between prime editing activity and cellular replication and disclose that the proportions between accurate prime editing events and unwanted byproducts can be influenced by the target-cell context. Hence, adenovector particles permit the efficacious delivery and testing of prime editing reagents in human cells independently of their transformation and replication statuses. The herein integrated gene delivery and gene editing technologies are expected to aid investigating the potential and limitations of prime editing in numerous experimental settings and, eventually, in ex vivo or in vivo therapeutic contexts.