Quality assurance of monoclonal products: Virologic and molecular biologic considerations

Summary The FDA has set limits concerning the viral and molecular contamination of monoclonal antibody products intended for human use. Industry has an obligation to be as familiar with these limits as it has been with federal requirements pertaining to pyrogens and bacteria. The assessment of risk from polynucleotides, based on molecular biologic and existing technical limitations, is discssed, as is the strategy of validating the purification of monoclonal antibodies of viral contaminants in terms of an indicator organism concept.     

Towards microbiological quality assurance in radiation sterilization processing: a limiting case model

A Limiting Case Model has been developed which describes the dependence on radiation dose of the proportion of items, in a population of items subjected to irradiation, which are contaminated by one or more organisms. This model is independent of the initial distribution of numbers of micro-organisms on items and represents a conservative approach to estimation of the proportions of non-sterile items in an irradiated population of items.     

A Bayesian mixture model relating dose to critical organs and functional complication in 3D conformal radiation therapy

A goal of cancer radiation therapy is to deliver maximum dose to the target tumor while minimizing complications due to irradiation of critical organs. Technological advances in 3D conformal radiation therapy has allowed great strides in realizing this goal; however, complications may still arise. Critical organs may be adjacent to tumors or in the path of the radiation beam. Several mathematical models have been proposed that describe the relationship between dose and observed functional complication; however, only a few published studies have successfully fit these models to data using modern statistical methods which make efficient use of the data. One complication following radiation therapy of head and neck cancers is the patient's inability to produce saliva. Xerostomia (dry mouth) leads to high susceptibility to oral infection and dental caries and is, in general, unpleasant and an annoyance. We present a dose-damage-injury model that subsumes any of the various mathematical models relating dose to damage. The model is a nonlinear, longitudinal mixed effects model where the outcome (saliva flow rate) is modeled as a mixture of a Dirac measure at zero and a gamma distribution whose mean is a function of time and dose. Bayesian methods are used to estimate the relationship between dose delivered to the parotid glands and the observational outcome-saliva flow rate. A summary measure of the dose-damage relationship is modeled and assessed by a Bayesian chi(2) test for goodness-of-fit.     

Towards microbiological quality assurance in radiation sterilization processing: the limiting case model applied to a microbial population having a distribution of radiation responses

A limiting case model, developed earlier to provide a conservative estimate of the proportion of non-sterile items ( P ) in an irradiated population of items contaminated with micro-organisms of a single type, has been extended to include items having organisms of different types. Estimates of P are compared with exact values derived on purely theoretical grounds and with values obtained by computer simulation of microbiological inactivation on items.     

Degradation of biochemical activity in soil sterilized by dry heat and gamma radiation.

The activities of three enzymes present in soil, phosphatases, urease, and decarboxylase, were monitered as indicators of the loss of biochemical information occurring when soil was sterilized by dry heat (0.08% relative humidity), gamma radiation, or a combination of both. More enzymatic activity was retained in soil sterilized by a long exposure to dry heat at relatively low temperature (8 weeks at 100.5 degrees C) than by a shorter exposure to a higher temperature (2 weeks at 124.5 degrees C). No enzymatic activity was detectable in soil sterilized by an even higher temperature (4 days at 148.5 degrees C). Soil sterilized with 7.5 Mrads of radiation retained much higher enzymatic activity than with heat sterilization. Combining sublethal doses of heat radiation effectively sterilized the soil and yielded enzymatic activities higher than those of soil sterilized by dry heat alone but lower than those of soil sterilized by radiation.     

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.

     

Intracranial pressure. II. Experimental verification of a mathematical model]

The authors report the results of experiments (performed on the dog in vitro and in vivo) which were designed to verify hypotheses made concerning vascular elasticity and characteristics of CSF resorption, as well as to test the validity of a mathematical model that was described in a previous paper. The authors concluded that the equations of the model account for most of the phenomena satisfactorily, but fail to predict a rebound phenomena which occurs after sudden injections in the cisterna magna. The elasticity coefficient S appears to be constant over a range of 200 mm Hg. On the other hand, the filtration coefficient K depends upon pressure, probably due to morphological changes in the size and shape of the CSF valves. These discrepancies are taken into account in a more general model that is presented in the following paper.     

Chromosome aberration frequency and radiation dose to lymphocytes by alpha-particles from internal deposit of Thorotrast

Summary Frequencies of chromosome aberrations in peripheral blood lymphocytes from 63 Thorotrast patients were analysed basing on the age distribution of lymphocytes. The frequency and distribution of chromosome aberrations among lymphocytes are best explained if we assume that the lymphocytes are renewed as an exponential function of time and spend most of their lifetime in the distributive pool where, while exchange of lymphocytes is taking place, the lymphocytes are hit by-particles from Thorotrast aggregates resulting in the formation of chromosome aberrations and killing at specific rates per hit. The model predicts that the aberration frequency is rather insensitive to the fluence rate because of modulation by cell killing by hit. Fitting the observed data to the model showed that approximately 0.8 dicentrics and rings were produced by a single path of-particle and average fluence rate to lymphocytes in a group of patients with the highest aberration frequency was estimated to be about 1.5 hits or 87 rad/lymphocyte/year.     

Molecular mechanisms of radiation carcinogenesis and the linear, non-threshold dose response model of radiation risk estimation

Recent research in molecular radiation carcinogenesis is reviewed with the specific aim of exploring the implications this research may have on the dose response relationship of radiation-induced cancer at low doses and low dose rates. It is concluded that the linear non-threshold dose response hypothesis may be used in radiation protection planning as a simple, convenient method to optimize procedures and regulations, but should not be mistaken as a stringent scientific conclusion directly derived from the present state of knowledge of the processes involved in radiation carcinogenesis.     

Quality assurance in cytopathology. Recommendations and ongoing quality assurance activities of the American Society of Clinical Pathologists

The recommendations and activities of the American Society of Clinical Pathologists (ASCP) pertaining to quality assurance in cytopathology are reviewed. To assure the early detection of cervical cancer, the ASCP recommends that all women who are, or have been, sexually active should have an annual Papanicolaou smear examination, the reasons for which are discussed. To assure the optimal quality in the evaluation of these smears, the ASCP recommends that the screening should be performed (1) in an accredited laboratory by certified cytotechnologists, (2) for whom realistic workloads have been established, (3) under the supervision of a pathologist adequately trained in cytology, (4) who will maintain an ongoing quality assurance program. In addition, (5) Papanicolaou smear evaluations should be available to all patients at a reasonable cost and (6) the financial incentives for the performance of low-quality screening should be eliminated. The society's quality assurance activities discussed include the Check Sample program, self-assessment examinations, meetings and publications; planned additions to this arena include a challenge exam and proficiency survey mechanisms.     

Physicochemical, toxicological and hygienic aspects of ethylene oxide application for the sterilization of medical products. III. Setting hygienic norms in sterilized medical products

Some of the drawbacks are discussed associated with hygienic norms for gaseous sterilizing agents in polymeric products for medical applications. A new approach is proposed for regulating ethylene oxide (EO) in such products. To this end, limiting types of biological effect and its pathways have been determined and substantiated as well as the reliability factor for establishing hygienic norms for EO in medical products. Daily threshold EO limit values were calculated for momentary and repeated exposure of humans. The duration of long-term and short-term exposure of the patient to sterilized products was evaluated on the basis of EO extraction kinetics from a variety of materials. Using daily threshold limit values, threshold residual values (TRV) of EO were calculated for different groups of polymeric products for medical applications.     

Multiple assembly states of lumazine synthase: a model relating catalytic function and molecular assembly

Lumazine synthases have been observed in the form of pentamers, dimers of pentamers, icosahedral capsids consisting of 60 subunits and larger capsids with unknown molecular structure. Here we describe the analysis of the assembly of native and mutant forms of lumazine synthases from Bacillus subtilis and Aquifex aeolicus at various pH values and in the presence of different buffers using small angle X-ray scattering and electron microscopy. Both wild-type lumazine synthases are able to form capsids with a diameter of roughly 160 A and larger capsids with diameters of around 300 A. The relative abundance of smaller and larger capsids is strongly dependent on buffer and pH. Both forms can co-exist and are in some cases accompanied by other incomplete or deformed capsids. Several mutants of the B. subtilis lumazine synthase, in which residues in or close to the active site were replaced, as well as an insertion mutant of A. aeolicus lumazine synthase form partially or exclusively larger capsids with a diameter of about 300 A. The mutations also reduce or inhibit enzymatic activity, suggesting that the catalytic function of the enzyme is tightly correlated with its quaternary structure. The data show that multiple assembly forms are a general feature of lumazine synthases.     

Health risks of low dose ionizing radiation in humans: a review

Radiobiologists have been struggling to estimate the health risks from low doses of radiation in humans for decades. Health risks involve not only neoplastic diseases but also somatic mutations that may contribute to other illnesses (including birth defects and ocular maladies) and heritable mutations that may increase the risk of diseases in future generations. Low dose radiation-induced cancer in humans depends on several variables, and most of these variables are not possible to correct for in any epidemiologic study. Some of the confounding factors include (i) interaction of radiation with other physical (UV light), chemical, and biological mutagens and carcinogens in a synergistic manner; (ii) variation in repair mechanisms that depend on dose; (iii) variation in sensitivity of bystander cells to subsequent radiation exposure that depends on whether they have been pre- or postirradiated; and (iv) variation in adaptive response that depends on radiation doses and protective substances (antioxidants). In our opinion, both the linear no-threshold-response and the threshold-response models might not be suitable in predicting cancer risk at low radiation doses in a quantitative sense. Low doses of ionizing radiation should not be considered insignificant for risks of somatic and heritable mutations and neoplastic and nonneoplastic diseases in humans.     

A theoretical model of the knee and ACL: theory and experimental verification.

A three-dimensional mathematical model of the human knee joint was developed to examine the role of single ligaments, such as an anterior cruciate ligament (ACL) graft in ACL reconstruction, on joint motion and tissue forces. The model is linear and valid for small motions about an equilibrium position. The knee joint is modeled as two rigid bodies (the femur and the tibia) interconnected by deformable structures, including the ACL or ACL graft, the cartilage layer, and the remainder of the knee tissues (modeled as a single element). The model was demonstrated for the equilibrium condition of the knee in extension with an anterior tibial force, causing anterior drawer and hyperextension. The knee stiffness matrix for this condition was measured for a human right knee in vitro. Predicted model response was compared with experimental observations. Qualitative agreement was found between model and experiment, validating the model and its assumptions. The model was then used to predict the change in graft and cartilage forces and joint motion of the knee due to an increment of load in the normal joint both after ACL removal and with various altered states simulating ACL reconstructions. Results illustrate the interdependence between loads in the ACL graft, other knee structures, and contact force. Stiffer grafts and smaller maximum unloaded length of the ligament lead to higher graft and contact forces. Changes in cartilage stiffness alter load sharing between ACL graft and other joint tissues.     

Investigations into the systemic production of aldehyde-derived peroxidation products in a murine model of acute iron poisoning: a dose response study

Acute iron poisoning remains a leading cause of morbidity and mortality in pre-school aged children in North America. Acute iron poisoning leads to organ damage, such as respiratory difficulties, cardiac arrhythmias, and possible death. The mechanism of iron toxicity is not fully understood, though it is thought that free iron is able to catalyze the production of harmful oxygen free radicals, which can damage all biochemical classes including lipid membranes, proteins, and DNA. Accordingly, we hypothesized that acute iron loading results in dose-dependent increases in oxygen free radical production, as quantified by the cytotoxic aldehydes hexanal, 4-hydroxynonenal, and malondialdehyde, in an experimental murine model. In support of our hypothesis, significant dose-dependent increases in all aldehydes investigated were reported in comparison to controls (p < 0.001). This murine model will assist in providing a better understanding of possible mechanism(s) of injury and organ dysfunction following acute iron poisoning, and for the development and evaluation of treatment regimes.     

Parallel production and verification of protein products using a novel high-throughput screening method

Protein production and analysis in a parallel fashion is today applied in laboratories worldwide and there is a great need to improve the techniques and systems used for this purpose. In order to save time and money, a fast and reliable screening method for analysis of protein production and also verification of the protein product is desired. Here, a micro-scale protocol for the parallel production and screening of 96 proteins in plate format is described. Protein capture was achieved using immobilized metal affinity chromatography and the product was verified using matrix-assisted laser desorption ionization time-of-flight MS. In order to obtain sufficiently high cell densities and product yield in the small-volume cultivations, the EnBase® cultivation technology was applied, which enables cultivation in as small volumes as 150 μL. Here, the efficiency of the method is demonstrated by producing 96 human, recombinant proteins, both in micro-scale and using a standard full-scale protocol and comparing the results in regard to both protein identity and sample purity. The results obtained are highly comparable to those acquired through employing standard full-scale purification protocols, thus validating this method as a successful initial screening step before protein production at a larger scale.