Impact of cell culture process changes on endogenous retrovirus expression

Cell culture process changes (e.g., changes in scale, medium formulation, operational conditions) and cell line changes are common during the development life cycle of a therapeutic protein. To ensure that the impact of such process changes on product quality and safety is minimal, it is standard practice to compare critical product quality and safety attributes before and after the changes. One potential concern introduced by cell culture process improvements is the possibility of increased endogenous retrovirus expression to a level above the clearance capability of the subsequent purification process. To address this, retrovirus expression was measured in scaled down and full production scaled Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes. Two highly sensitive, quantitative (Q)-PCR-based assays were used to measure endogenous retroviruses. It is shown that cell culture process changes that primarily alter media components, nutrient feed volume, seed density, cell bank source (i.e., master cell bank vs. working cell bank), and vial size, or culture scale, singly or in combination, do not impact the rate of retrovirus expression to an extent greater than the variability of the Q-PCR assays (0.2-0.5 log(10)). Cell culture changes that significantly alter the metabolic state of the cells and/or rates of protein expression (e.g., pH and temperature shifts, NaButyrate addition) measurably impact the rate of retrovirus synthesis (up to 2 log(10)). The greatest degree of variation in endogenous retrovirus expression was observed between individual cell lines (up to 3 log(10)). These data support the practice of measuring endogenous retrovirus output for each new cell line introduced into manufacturing or after process changes that significantly increase product-specific productivity or alter the metabolic state, but suggest that reassessment of retrovirus expression after other process changes may be unnecessary.     

Allele frequencies of apolipoprotein E gene polymorphisms in the protein coding region and promoter region (-491A/T) in a healthy Norwegian population

This study examines the distribution of apolipoprotein E (APOE) alleles in a population of healthy male and female Norwegians (n = 798) below the age of 40. The -491A/T polymorphism of the promoter region of the APOE gene was also examined. A seminested polymerase chain reaction was applied in the genotyping. The results showed that the E3 allele had the highest frequency (0.744), followed by E4 (0.198) and E2 (0.058). The APOE frequencies found in this study differ significantly from those obtained in earlier Norwegian APOE phenotypings. The allele frequencies in the -491 site of the promoter region were 0.845 for the A allele and 0.155 for the T allele. The genotype frequency was highest for AA (0.707), followed by AT (0.277) and TT (0.016). Moreover, the A allele was in linkage disequilibrium to E4.     

Hippocampal neurons responding to first-time dislocation of a target object

To examine how hippocampal neurons respond to a mismatch between retrieved and actual experience, we trained rats to find a hidden platform at a particular location in an annular watermaze and then moved the platform. Several cells that were silent at the new platform location before the move fired vigorously when the rat found the goal. The new activity was paralleled by reduced discharge in a subset of simultaneously recorded interneurons. The pattern of activity returned toward its original configuration as the rat learned the new location. The activation of specific hippocampal neurons following dislocation of a target object may be essential for synaptic plasticity and adaptive modification of the animal's representation of the environment.     

Evaluation of a chiral stationary phase derived from a simple Diels-Alder reaction

The bicyclic, C(2)-symmetric dicarboxylic acid obtained from the cycloaddition of fumaric acid to anthracene can readily be prepared in enantiomerically pure form on a large scale. Conversion of either enantiomer of the diacid into its corresponding bis-allylamide yields a selector unit, used as a building block in the synthesis of a chiral stationary phase (CSP). The terminal C-C double bonds in the selector unit were used in a hydrosilylation reaction involving a multifunctional hydrosilane to effect polymerization, crosslinking, and immobilization to the vinyl-silica used as support. A study of the enantiomer-discriminating abilities of the new CSP was made. Large separation factors were obtained for a series of benzodiazepinones, which were studied in further detail by the use of different retention modifiers. To some extent, rationalization of retention data has been achieved in terms of competition for hydrogen bonding.     

Humic substances in drinking water and the epidemiology of thyroid disease

Thyroid diseases are common in all populations but the type and frequency depends on environmental factors. In Denmark geographical differences in iodine intake are caused by different iodine contents of drinking water, which varies from < 1 to 139 microg iodine per litre. Comparative epidemiologic studies have demonstrated considerable differences in type and occurrence of thyroid disease with more goitre and hyperthyroidism in Aalborg with water iodine content around 5 microg/L, and more hypothyroidism in Copenhagen with water iodine around 20 microg/L. In Denmark, iodine in ground water is bound in humic substances, which have probably leached from marine sediments in the aquifers. Interestingly, humic substances in water from other parts of the world have goitrogenic properties, especially humic substances from coal and shale. Humic substances are heterogeneous mixtures of naturally occurring molecules, produced by decomposition of plant and animal tissues. The effect of humic substances in drinking water on the epidemiology of thyroid disease probably depends on the source of aquifer sediments.     

Physiological imaging of the lung: single-photon-emission computed tomography (SPECT).

Emission tomography provides three-dimensional, quantitative images of the distribution of radiotracers used to mark physiological, metabolic, or pathological processes. Quantitative single photon emission computed tomography (SPECT) requires correction for the image-degrading effects due to photon attenuation and scatter. Phantom experiments have shown that radioactive concentrations can be assessed within some percentage of the true value when relevant corrections are applied. SPECT is widely spread, and radiotracers are available that are easy to use and comparably inexpensive. Compared with other methods, SPECT suffers from a lower spatial resolution, and the time required for image acquisition is longer than for some alternative methods. In contrast to some other methods, SPECT allows simultaneous imaging of more than one process, e.g., both regional blood flow and ventilation, for the whole lung. SPECT has been used to explore the influence of posture and clinical interventions on the spatial distribution of lung blood flow and ventilation. Lung blood flow is typically imaged using macroaggregates of albumin. Both radioactive gases and particulate aerosols labeled with radioactivity have been used for imaging of regional ventilation. However, all radiotracers are not equally suited for quantitative measurements; all have specific advantages and limitations. With SPECT, both blood flow and ventilation can be marked with radiotracers that remain fixed in the lung tissue, which allows tracer administration during conditions different from those at image registration. All SPECT methods have specific features that result from the used radiotracer, the manner in which it is administered, and how images are registered and analyzed.     

Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO‐cell derived biotherapeutics

During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non‐electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science‐based process validation strategies to ensure viral safety of biotechnology products. Biotechnol. Bioeng. 2009; 104: 371–380 © 2009 Wiley Periodicals, Inc.     

Binding Affinity,Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200

The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovarian cancer with ²¹¹At-Rebmab200. Here, the biodistribution of ²¹¹At-Rebmab200 was evaluated, as was the utility of ^99mTc-Rebmab200 for bioimaging. Rebmab200 was directly compared with its murine counterpart MX35 in terms of its in-vitro capacity for binding the immobilized NaPi2B epitope and live cells; we also assessed its biodistribution in nude mice carrying subcutaneous OVCAR-3 tumors. Tumor antigen and cell binding were similar between Rebmab200 and murine MX35, as was biodistribution, including normal tissue uptake and in-vivo tumor binding. We also demonstrated that ^99mTc-Rebmab200 can be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics.     

Time Trends in Incidence and Mortality of Acute Myocardial Infarction,and All-Cause Mortality following a Cardiovascular Prevention Program in Sweden

Background

In 1988, a cardiovascular prevention program which combined an individual and a population-based strategy was launched within primary health-care in Sollentuna, a municipality in Stockholm County. The aim of this study was to investigate time trends in the incidence of and mortality from acute myocardial infarction and all-cause mortality in Sollentuna compared with the rest of Stockholm County during a period of two decades following the implementation of a cardiovascular prevention program.

Materials and Methods

The average population in Sollentuna was 56,589 (49% men) and in Stockholm County (Sollentuna included) 1,795,504 (49% men) during the study period of 1987–2010. Cases of hospitalized acute myocardial infarction and death were obtained for the population of Sollentuna and the rest of Stockholm County using national registries of hospital discharges and deaths. Acute myocardial infarction incidence and mortality were estimated using the average population of Sollentuna and Stockholm in 1987–2010.

Results

During the observation period, the incidence of acute myocardial infarction decreased more in Sollentuna compared with the rest of Stockholm County in women (-22% vs. -7%; for difference in slope <0.05). There was a trend towards a greater decline in Sollentuna compared to the rest of Stockholm County in the incidence of acute myocardial infarction (in men), acute myocardial mortality, and all-cause mortality but the differences were not significant.

Conclusion

During a period of steep decline in acute myocardial infarction incidence and mortality in Stockholm County the municipality of Sollentuna showed a stronger trend in women possibly compatible with favorable influence of a cardiovascular prevention program.

Trial Registration

ClinicalTrials.gov NCT02212145