Good Manufacturing Procedures Research Articles

Comment on “Quality evaluation of microscopy and scanned histological images for diagnostic purposes”: Are scanners better than microscopes?

Comment on “Quality evaluation of microscopy and scanned histological images for diagnostic purposes”: Are scanners better than microscopes?

Adoptive transfer of virus-directed T cells: will this fly for flu?

Adoptive transfer of virus-specifi c T cells has been used successfully to prevent and treat severe virus infections in stem cell transplant recipients. In this issue of Cytotherapy, Gaundar et al. (1) describe the possibility of extending the application to infl uenza. Cytomegalovirus (CMV), Epstein–Barr virus (EBV) and adenovirus are three agents that commonly produce morbidity and mortality in immunocompromised patients after stem cell transplantation. All have been controlled effectively by adoptively transferring virus-specifi c T cells generated from donor T lymphocytes by ex vivo culture with live virus or viral antigens (2–4). Protection by these T cells appears to be long lived and mediated by both CD4 and CD8 effector T cells. Gaundar et al. (1) have generated infl uenza virusspecifi c effector T cells using dendritic cells pulsed with commercial inactivated trivalent infl uenza vaccine, a component that will facilitate translation to a clinical protocol. However, a number of potential concerns are introduced by the biology of infl uenza virus and the design of the vaccine. Unlike CMV and EBV, infl uenza is a genetically unstable virus, susceptible to both antigenic drift, involving amino acid changes in immunogenic domains, and genetic shift, involving reassortment of its eight RNA during co-infection of a single cell by two different subtypes (5,6). Such reassortant viruses containing novel combinations of RNA are thought to be the source of major infl uenza pandemics. Vaccines are designed to protect against newly emergent strains and subtypes but elicit non-cross-reactive neutralizing antibodies to the viral hemagglutinin and neuraminidase and are not formulated to elicit T-cell responses in vivo. T cells have the benefi t of recognizing conserved epitopes in viruses, so that natural infection with infl uenza has long been known to induce crossreactive T cells (7–9) that can protect against unrelated subtypes (10). Animal studies have shown that infection with live or live-attenuated viruses are protective or partially protective against multiple strains. However, it is unclear whether virus-specifi c T cells generated in vitro using inactivated viruses would elicit T cells that effectively recognize infections with different infl uenza strains and subtypes. If not, the complexity of any adoptive cell-mediated therapy would greatly increase. Moreover, it is not clear whether T cells alone, even if cross-reactive, will be suffi cient to prevent or clear infl uenza virus in the absence of the strain-specifi c antibodies. Some of these issues are addressed in the current study (1). The investigators pulsed autologous dendritic cells with the trivalent vaccine Fluvax (CSL Laboratories), which contains inactivated A/ California/7/2009-H1N1, A/Wisconsin/15/2009H3N2 and B/Brisbane/60/2008 viruses, and obtained a substantial expansion of polyfunctional infl uenzaspecifi c CD4 and CD8 T cells. These T cells were reactive with all three viruses and had phenotypes indicative of both cytotoxic effector function and the ability to help antibody production. As the T-cell manufacturing process was good manufacturing procedure (GMP)-compliant, in principle these cells could be transferred to stem cell transplant recipients at high risk for disease or suffering from infl uenza infections. Although their results are encouraging, several major questions remain unanswered. For example, we do not yet know whether Fluvax-activated T cells can recognize unrelated strains and subtypes, or if the T-cell donors must be vaccinated or naturally infected Cytotherapy, 2012; 14: 133–134

QUALIDADE NA PRODUÇÃO DE REFEIÇÕES DE UMA UNIDADE DE ALIMENTAÇÃO E NUTRIÇÃO (UAN)

This study aimed to evaluate the food production and then act to increase the quality and security of a service, in order to meet the requirements for qualification HAPPC, using good manufacturing procedures as starting point for implementation. It is characterized as an intervention study, emphasizing the quality of foods and the use of good manufacturing procedures at the foodservice and nutrition units, located in Botucatu-SP. It was composed of 2 moments. In the first, we applied a check-list for ascertainment of non-conformity and in the second moment a training “in loco” was given for the collaborators. The lack of hygienic cleaning of hands is seen as an important negative point. We verified that the biggest difficulties and irregularities are related to the manipulators which don´t follow the rules. Good manufacturing procedures were obtained to increase the quality and the security of foods in the unit, but these must be kept constantly adjusted to the standards.

Positive Selection and Expansion of Cytomegalovirus-specific CD4 and CD8 T Cells in Sealed Systems

Administration of pathogen-specific T-cell lines can reconstitute the cellular immune function of immunocompromised patients. Selection and expansion of specific T cells for reinfusion pose unique challenges owing to the fact that good manufacturing procedures must be implemented. Cytokine secretion-based methods can identify and select specific T cells. We showed here that it is possible to combine this method with procedures for cell handling performed in a sealed, unbreached system from start to end. Peripheral blood mononuclear cells, obtained from blood samples and processed in a sealed system, were stimulated in Teflon bags with a library of selected CD4 and CD8 peptides derived from the immunodominant cytomegalovirus protein pp65. The stimulated T cells were labeled with reagents for interferon-gamma surface capture and selected on a magnetic column using a sealed system connected to the Teflon bags. Elution and final expansion were also performed with an unbreached protocol with preservation of sterility even if the steps were run on the bench top. Expanded cells exhibited the appropriate functions. The use of this unbreached procedure proves that safety of cellular products generated in a good manufacturing procedures facility can be further improved. Similar sealed protocols can also be applied for T-cell therapies directed against tumor antigens.

Phase I Clinical Trial of Autologous Ascites-derived Exosomes Combined With GM-CSF for Colorectal Cancer

Exosomes are small membrane vesicles that are secreted by a multitude of cell types. The exosomes derived from dendritic cells (Dex), tumor cells (Tex), and malignant effusions demonstrate immunomodulatory functions, and are even under clinical trial for cancer treatments. In this study we report the phase I clinical trial of the ascites-derived exosomes (Aex) in combination with the granulocyte–macrophage colony-stimulating factor (GM-CSF) in the immunotherapy of colorectal cancer (CRC). The Aex isolated by sucrose/D2O density gradient ultracentrifugation are 60–90-nm vesicles that contain the diverse immunomodulatory markers of exosomes and tumor-associated carcinoembryonic antigen (CEA). Totally 40 patients (HLA-A0201+CEA+) with advanced CRC were enrolled in the study, and randomly assigned to treatments with Aex alone or Aex plus GM-CSF. Patients in both groups received a total of four subcutaneous immunizations at weekly intervals. We found that both therapies were safe and well tolerated, and that Aex plus GM-CSF but not Aex alone can induce beneficial tumor-specific antitumor cytotoxic T lymphocyte (CTL) response. Therefore, our study suggests that the immunotherapy of CRC with Aex in combination with GM-CSF is feasible and safe, and thus can serve as an alternative choice in the immunotherapy of advanced CRC.

A sealed and unbreached system for purification, stimulation, and expansion of cytomegalovirus‐specific human CD4 and CD8 T lymphocytes

Recent clinical trials have demonstrated the efficacy of adoptive cellular therapy with virus-specific lymphocytes in patients with defective cellular immune responses. Immunoreconstitution has become a challenge for cellular immunology and for transfusion medicine. In fact, both expertises are required to provide effective and safe cellular products. Because of in vitro manipulation, T-lymphocyte cultures are at risk of contamination even under good manufacturing procedure (GMP) conditions. To further improve the quality of these GMP cellular products, a procedure was designed for purification, stimulation, and expansion of antigen-specific CD4 and CD8 T-lymphocytes in a sealed, unbreached system. Leukopacks from the blood bank that fulfill the requirements of a GMP product were the starting material. Gradient separation and washing were performed in bags with sterile connecting devices on the bench-top, as well as addition of ingredients (antigen, interleukin-2) or transfer to larger bags. The method is described in detail, and it is shown that increase in number of cytomegalovirus-specific CD4 or CD8 T-lymphocytes was similar to procedures based on open culture systems. Cell expansion after 4 weeks ranged from 800- to 2400-fold for CD4 lymphocytes and 300- to 900-fold for CD8 lymphocytes. Antigen specificity and loss of alloreactivity were demonstrated on the expanded cells with proliferation, intracytoplasmic interferon gamma-gamma staining, cytolytic activity, and pentamer binding. This procedure can be applied to improve sterility under GMP conditions when T-cell lines are generated for adoptive immunotherapy and may increase biosafety for the staff when cell lines are generated from subjects infected with dangerous pathogens.

Risks and Benefits of Supplement Use

Although dietary supplements offer consumers the ability to participate in their own health and well being, steps need to be taken to educate the public and clinicians to prevent misuse and encourage proper use. Better patient education through health care providers and the establishment of good manufacturing procedures for supplement producers will benefit those wishing to take dietary supplements. Currently, supplement users may be exposed to increased risk due to the lack of required good manufacturing procedures, poor communication between patients and health care providers, and the manner in which dietary supplements can be marketed without proof of safety or efficacy derived from large clinical trials.

Adequação das boas práticas de fabricação em serviços de alimentação

OBJETIVO: Classificar 50 estabelecimentos produtores de alimentos, da região de Brasília, Distrito Federal, de acordo com o cumprimento dos itens imprescindíveis do formulário publicado pela Agência Nacional de Vigilância Sanitária, a fim de avaliar as boas práticas da fabricação de alimentos. MÉTODOS: Neste estudo empreendeu-se metanálise de dois trabalhos monográficos sobre estabelecimentos comerciais produtores de alimentos (hotéis e restaurantes), associados a uma amostra pesquisada por esses autores, dividida em três blocos: restaurantes, unidades de alimentação e nutrição e hotéis, para comparação da adequação dos itens presentes nos formulários da Agência Nacional de Vigilância Sanitária, os quais, de acordo com as normas estabelecidas pela Agência, analisavam os itens: edificação, equipamentos, manipuladores, fluxo de produção e disponibilidade do manual de boas práticas na produção de alimentos. Ainda de acordo com o estipulado pela Agência Nacional de Vigilância Sanitária, os estabelecimentos foram classificados em três grupos, segundo o cumprimento percentual dos itens imprescindíveis constantes do formulário: o grupo I, com mais de 70,0% de cumprimento dos itens; o grupo II, com 30,0% a 69,9% de cumprimento; e o grupo III, cumprindo menos de 30,0% dos itens imprescindíveis. RESULTADOS: As análises realizadas revelaram que todas as unidades hoteleiras foram classificadas no grupo II. Os restaurantes comerciais foram classificados no grupo II (33,3%) e no grupo III (66,7%). Do total de unidades de alimentação e nutrição avaliadas, 80,0% foram classificadas no grupo II e 20,0% no grupo I. CONCLUSÃO: Os achados demonstraram que as unidades de alimentação e nutrição foram mais bem classificadas nos itens equipamentos e existência do Manual de Boas Práticas. Os hotéis destacaram-se nos itens edificação, manipulação e fluxo de produção. Os restaurantes comerciais obtiveram o pior resultado quando comparados às demais unidades analisadas.

HACCP certification of food services in Philippine inter-island passenger vessels

This paper presents the efforts of the Bureau of Quarantine and International Health Surveillance (BQIHS), Department of Health (DOH), Philippines to launch a Good Manufacturing Procedure (GMP)–Hazard Analysis Critical Control Point (HACCP) Certification Program for food services operating in Philippine inter-island water vessels. The BQIHS improved its food sanitation audit system for inter-island ships by including in its traditional walk-through evaluation of GMP the review of documents for both GMP and HACCP, and the verification of HACCP implementation. The BQIHS is now identified as a GMP–HACCP certification provider to specific sectors of the local food service industry on a national level.

Cell transplantation in heart failure management

Heart failure is becoming a major issue for public health in western countries and the effect of currently available therapies is limited. Therefore cell transplantation was developed as an alternative strategy to improve cardiac structure and function. This review describes the multiple cell types and clinical trials considered for use in this indication. Most studies have been developed in models of post-ischemic heart failure. The transplantation of fetal or neonatal cardiomyocytes has proven to be functionally successful, but ethical as well as immunological and technical reasons make their clinical use limited. Recent reports, however, suggested that adult autologous cardiomyocytes could be prepared from stem cells present in various tissues (bone marrow, vessels, adult heart itself, adipose tissue). Alternatively, endothelial progenitors originating from bone marrow or peripheral blood could promote the neoangiogenesis within the scar tissue. Hematopietic stem cells prepared from bone marrow or peripheral blood have been proposed but their differentiation ability seems limited. Finally, the transplantation of skeletal muscle cells (myoblasts) in the infarcted area improved myocardial function, in correlation with the development of skeletal muscle tissue in various animal models. The latter results paved the way for the development of a first phase I clinical trial of myoblast transplantation in patients with severe post-ischemic heart failure. It required the scale-up of human cell production according to good manufacturing procedures, started in june 2000 in Paris and was terminated in november 2001, and was followed by several others. The results were encouraging and prompted the onset of a blinded, multicentric phase II clinical trial for skeletal muscle cells transplantation. Meanwhile, phase I clinical trials also evaluate the safeness and efficacy of various cell types originating from the bone marrow or the peripheral blood. However, potential side effects related to the biological properties of the cells or the delivery procedures are being reported. High quality clinical trials supported by strong pre-clinical data will help to evaluate the role of cell therapy as a potential treatment for heart failure.

Efficient transduction and selection of human T-lymphocytes with bicistronic Thy1/HSV1-TK retroviral vector produced by a human packaging cell line.

T-cells expressing the HSV1-TK suicide gene can be used for the control of graft-versus-host disease following allogeneic stem cell transplantation. To develop clinical trials based on such a strategy, we have generated under good manufacturing procedures a novel 'split genome' human packaging cell line (1704 cells). To minimize the risk of generating replication-competent retroviruses, pol was truncated to remove sequences overlapping with env. To improve retroviral infection and selection of transduced T-cells, high titers of GALV-pseudotyped retroviral particles harboring a bicistronic Thy1-IRES-TK vector coding for the CD90 GPI-anchored membrane molecule were produced by 1704 cells. Using 1704 cell supernatant and an optimized transduction protocol, approximately 50% of primary T-cells were transduced and could then be purified (approximately 95%) using clinical-grade immunomagnetic beads directed against CD90. Over 96% of these OKT3/IL-2-activated CD90(+)-selected T-cells were killed by ganciclovir. Cell proliferation and cytokine production of transduced T-cells and HLA-restricted cytotoxicity of transduced T-cell clones were identical to those of their non-transduced counterparts cultured under the same conditions. GALV-pseudotyped retroviral particles harboring a bicistronic Thy1-IRES-TK vector allow efficient transduction and rapid selection of human T-cells under conditions applicable for clinical trials using the new human 1704 packaging cell line.

Microbiology of Kwek-Kwek: An Emerging Philippine Streetfood

The microbiology of kwek-kwek as a Philippine emerging streetfood made from boiled quail eggs, which have been battered and fried was studied. The process of water-cooling and manual peeling of the boiled shell eggs were shown to increase the TPCs, and coliform and staphylococci counts of the product. The batter was also shown to contribute to microbial contamination of the product. The microbial profile of ready-to-eat kwek-kwek during vending was: 104-106 cfu/g TPC; ≤102 MPN/g coliform count; ≤104 MPN/g coagulase-positive staphylococci count; and presence of Salmonella sp. per 25 g sample. The flour-based sauces of kwek-kwek were found to contain higher microbial contaminants than the vinegar-based sauce. It was recommended that good manufacturing procedures within the boundary of streetfood trade economics be fully considered in the preparation and vending of the product.

High-level expression of the malaria blood-stage vaccine candidate Plasmodium falciparum apical membrane antigen 1 and induction of antibodies that inhibit erythrocyte invasion.

Apical membrane antigen 1 (AMA-1) is a highly promising malaria blood-stage vaccine candidate that has induced protection in rodent and nonhuman primate models of malaria. Authentic conformation of the protein appears to be essential for the induction of parasite-inhibitory antibody responses. Here we have developed a synthetic gene with adapted codon usage to allow expression of Plasmodium falciparum FVO strain AMA-1 (PfAMA-1) in Pichia pastoris. In addition, potential N-glycosylation sites were changed, exploiting the lack of conservation of these sites in Plasmodium, to obtain high-level secretion of a homogeneous product, suitable for scale-up according to current good manufacturing procedures. Purified PfAMA-1 displayed authentic antigenic properties, indicating that the amino acid changes had no deleterious effect on the conformation of the protein. High-titer antibodies, raised in rabbits, reacted strongly with homologous and heterologous P. falciparum by immunofluorescence. In addition, purified immunoglobulin G from immunized animals strongly inhibited invasion of red blood cells by homologous and, to a somewhat lesser extent, heterologous P. falciparum.

Food safety knowledge and practices of streetfood vendors in a Philippines university campus

A survey on food safety knowledge and practices of streetfood vendors from a representative urban university campus in Quezon City, Philippines was done. A face-to-face interview was conducted using a standardized survey tool containing 70 questions, which included queries on demographics and food safety knowledge and practices of streetfood vendors. Topics on food safety assessment in both practices and knowledge included: health and personal hygiene, good manufacturing procedures, food contamination, waste management, and food legislation. The study found that among the 54 streetfood vendors surveyed, knowledge on food safety concepts was established particularly on topics that dealt with health and personal hygiene, food contamination and good manufacturing procedures. However, vendors were shown to be not too knowledgeable in terms of food legislation and waste management. A significant gap between knowledge and practice on these topics was established and it was primarily attributed to the tendencies of street food vendors to compromise food safety for financial issues. Confusion in food legislation was established in this test microcosm because the purveyor of food safety regulations was not the local government health unit but the business concession office of the campus administration. The provision of continuous food safety education, some financial assistance through social services affiliations, and basic water and waste management utilities were recommended to diminish the gap between knowledge and practices of safe streetfood vending in school campuses.

An Update on the Therapeutic Orphan

Thirty years ago, Dr Harry Shirkey recognized the serious dilemma of pediatric drug labeling, called therapeutic orphan, to capture its concept, and described remedial measures to minimize the dilemma. This review offers a brief exposition of the therapeutic orphan's arduous journey toward the goal of labeling of all drugs used for children. Aside from legal and US Food and Drug Administration (FDA) policy requirements to secure new drug application approval to market a drug, some may question reasons for a pediatric drug label, especially if a label exists for adult use and pediatric use information is distributed in literature reports. Major pragmatic reasons for a pediatric label are presented in Table 1. A label testifies to carefully reviewed clinical trial data that offer substantial evidence to support pediatric indications for which the drug has approved use. Perhaps of unappreciated importance is availability of information on the label. Time-consuming literature review and analysis often are not necessary when a pediatric label exists. Label information is compiled for the pediatric drug formulation, and it is this formulation that is approved. Literature reports often contain one or more formulations prepared by the investigator, and good manufacturing procedures or bioavailability estimates may be less than desired. A formulation on the label frequently has been evaluated for characteristics acceptable to children and hence compliance is strengthened. The drug, having met pediatric labeling requirements, is available for children and obviates the awkward double standard—one for adults (label necessary for marketing) and one for children (pediatric label not necessary for marketing or use). To the lay public, this pediatric labeling allows third-party reimbursement of drug costs otherwise denied. This relieves young parents of a truly indefensible economic burden, which in some cases has denied drug treatment to children. Add to this the refusal of some malpractice … Address correspondence to John T. Wilson, MD, Section on Clinical Pharmacology, Department of Pediatrics, Louisiana State University Medical Center, Shreveport, LA 71130.

Adverse effects of intravenous immunoglobulin therapy.

A growing body of literature documents that intravenous immunoglobulin prophylaxis and therapy is becoming applied to a steadily growing list of new indications. Some of these new indications have led to the use of intravenous immunoglobulin therapy in doctors offices, far from the hospital environment. Being stable products purified from blood or plasma donations, intravenous immunoglobulins must be considered as biological products in addition to their status as pharmaceutical products. This makes the study of adverse reactions reach beyond a mere drug safety surveillance programme into the realms of good manufacturing procedures guaranteeing not only intravenous tolerance but also sterility with regard to transfusion transmitted agents. The initially perceived adverse effects, stemming from complement activating aggregated immunoglobulin G, had the effect of slowing down widespread introduction of intravenous immunoglobulin therapy in the late 1970s. These adverse effects have now been eliminated with amendment of the appropriate manufacturing steps. However, new adverse effects, such as hyperviscosity, aseptic meningitis or renal insufficiency, have been observed which can be assigned to certain comnpounds of intravenous immunoglobulin, to administration regimens or to special patient characteristics. Adverse effects can be divided into 3 types: immediate adverse effects (those that occur during the infusion, e.g. anaphylactoid reactions); delayed adverse effects (those that occur hours to days after initiation of the infusion, e.g. renal, pulmonary, dermatological adverse effects, hyperviscosity, aseptic meningitis, arthritis, cerebral infarction, haemolysis and leucopenia) and; late adverse effects (e.g. transmission of infectious agents). We conclude from our analysis, that in general, intravenous immunoglobulin may be considered a well tolerated medical agent provided the indication for use is chosen carefully and use is monitored by a physician familiar with contraindications, risks, adverse effects and their appropriate management.