Transcriptomic profiling as a tool to characterize CHO cell processes

Global healthcare demands for affordable and accessible biological medicines are rapidly increasing as a result of an increasing number of patients. Biosimilars products have a potential to help decrease the price and increase the availability of the biological medicines. Monoclonal antibodies are a special class of biopharmaceutical proteins that are used to treat life-threatening diseases like cancer. Mammalian cells and more specific Chinese Hamster Ovary cells are the main production platform for these monoclonal antibodies. Many of the first products, which were developed in the early 1990s are going off patent opening the possibility to develop biosimilars. Nowadays, for the development of biosimilars and also completely new biopharmaceuticals it is required for pharmaceutical companies to include (aspects of) Quality by Design (QbD). This means that they need to have a good scientific understanding of the relation between critical process parameters (CPPs) and critical quality attributes (CQAs) of the products as wells as with key performance indicators (KPIs), like the cell concentration. These relations are usually statistical relations that are not based on detailed knowledge of biological mechanisms, although they are a good basis to acquire such knowledge. Transcriptomics is a state of the art technique to measure the global gene expression of a cell population and can give insight into the biological mechanisms that are behind the relation between CPPs, CQAs and KPIs. Furthermore, the transcriptome is a good representation of the physiological state of the cells and can thus be used for comparison of for example reactors of different scales, different processes, or comparison of a new bioreactor production run to past runs. The aim of this thesis is to study the value of transcriptome analysis for process development and the quality by design approach for the upstream cultivation process. All experiments in this thesis use the same CHO cell clone producing a monoclonal antibody. All transcriptome measurements are done using commercially available Affymetrix CHO Gene 2.1 ST arrays. Early process development for biopharmaceuticals is often partly done in small scale systems in a high throughput approach. Transcriptome analysis is used in this thesis to assess whether the small scale systems are representative for the production scale reactor. The focus of Chapter 2 is to have a better understanding of the differences in cell performance between uncontrolled (shake flask) and controlled (bioreactor) systems. In this chapter, we evaluated differences in gene expression profiles between shake flask and bioreactor cultures at three different time points during the exponential and stationary phase of a batch cultivation. Both systems showed a similar large variation in gene expression over time, meaning the differences between two cultivation systems are small. However, the small gene expression difference between the two systems occurred during a short period of cultivation during batch cultivation and could be directly linked to the absence of control of some of the process cultivation parameters such as pH and DO in the shake flask. For fed-batch processes larger differences are expected to be present between controlled and uncontrolled systems as compared to batch due to the higher cell densities, bolus addition of concentrated feeds and longer process duration. Therefore, we investigate in Chapter 3, the differences in gene expression between, shake flasks and 1 L bioreactors for a fed-batch cultivation process. The shake flask cultures grew faster than the bioreactor cultures and went earlier into the stationary and death phase. Using PCA the difference in time development was represented in PC1, while also a time independent difference was observed and represented by PC2. Although transcriptome data can also identify differentially expressed pathways, which may lead to the root cause of the differences, here we were not able to identify this root cause. In Chapter 4, transcriptome analysis was used to evaluate the down scale of a CHO cell fed-batch process from a 10 L bioreactor to an ambr 15® (ambr) system. In this case both systems have control of pH and DO. The results of this comparative transcriptomic study showed that the variation in gene expression was less than 6% based on PCA. Moreover, the gene and pathway analysis did not reveal a direct relation of this difference with scale differences or specific process conditions. To be able to obtain an objective meaning to this 6% difference more transcriptome experiments need to be done. In this Chapter we also show that differences in gene expression in the preculture quickly disappear in the culture systems, showing that the preculture did not have an effect on the comparison. In Chapter 5, we explore the use of transcriptomic profiling for monitoring long term production campaigns such as occur using a perfusion cultivation mode. The results show that transcriptome data can be linked to specific phases of a perfusion process like a period characterized by a cell size increase and a period with a specific nutrient limitation. More importantly, quantitative transcriptome data visualized using PCA. show a clear separation between the steady state data points and the other data points and can thus be used to identify the steady state. In Chapter 6, transcriptomic profiling as a tool for quality by design in the cultivation process of antibody biosimilars is discussed. Comparative transcriptomic analysis could play a role in validation of the scale down system by demonstrating similar responses of biological processes for both systems. Likewise it can be an useful tool to detect process deviations, evaluate process modifications, and process characterization. Furthermore, in combination with other analyses it can give insight into biological processes that link CCPs to CQAs. Thus, transcriptomic profiling can be part of process performance indicators and combined with other bioanalytical measure give a better understanding and faster development of the design space. When the analysis can be done routinely and sufficient fast it can possibly also be used at line to evaluate and control running production runs.</p

Insomnia in chronic renal patients on dialysis in Saudi Arabia

<p>Abstract</p> <p>Background</p> <p>Studies have shown that insomnia is a common sleep disorder among patients with end-stage renal disease (ESRD). This study aimed to assess the prevalence of insomnia in Saudi patients with ESRD who are on maintenance dialysis.</p> <p>Methods</p> <p>This was an observational cross-sectional study carried out over a period of five months in two hemodialysis centers in Saudi Arabia. To assess the prevalence of insomnia, we used the ICSD-2 definition. We also examined the association between insomnia and other sleep disorders, the underlying causes of renal failure, dialysis duration, dialysis shift, and other demographic data.</p> <p>Results</p> <p>Out of 227 enrolled patients, insomnia was reported by 60.8%. The mean patient age was 55.7 ± 17.2 years; 53.7% were male and 46.3% were female. Insomnia was significantly associated with female gender, afternoon hemodialysis, Restless Legs Syndrome, high risk for obstructive Sleep Apnea Syndrome and excessive daytime sleepiness (<it><b>P-values: </b></it>0.05, 0.01, < 0.0001, < 0.0001, and < 0.0001, respectively). No significant association was found between insomnia and other variables, including BMI, smoking habits, underlying etiology of renal failure, dialysis duration, association with hemoglobin, ferritin, and phosphorus or dialysis adequacy as measured by the Kt/V index.</p> <p>Conclusion</p> <p>Insomnia is common in dialysis patients and was significantly associated with other sleep disorders. Greater attention needs to be given to the care of dialysis patients with regard to the diagnosis and management of insomnia and associated sleep disorders.</p

Blood pressure control, lifestyle and disease awareness of Saudi hypertensive patients

Mortality and morbidity from hypertension have reached epidemic proportion worldwide. It has been estimated that 874 million adults globally have systolic blood pressure (SBP) of 140 mm Hg or higher. A recent study from Saudi Arabia found 15.2% of adult Saudis were hypertensive of whom 57.8% unaware of this diagnosis. We aim to evaluate the lifestyle advices given to Saudi hypertensive patients, their current lifestyle to determine the effects of these factors on their BP control. Nonrandom convenience sampling of Saudi patients followed up in the clinic by cross-sectional questionnaire. Their BP, blood sugar, and other anthropometric data were measured and provided self-filled questionnaire. Of all participants, 148 known hypertensives on treatment were included in the study with a mean age of 45.7 ± 29.0 years. The mean SBP and diastolic BP were 134.7 ± 21.4 and 85.0 ± 18.9 mm Hg, respectively. The overall awareness score was 77.5% with the highest awareness score for “BP can be controlled by proper management” (93.2%) and the lowest score given for “BP is not affected by alcohol consumption” (63.4%). We found significantly lower mean SBP in those with higher awareness in five of the nine awareness areas inquired. We believe that educating hypertensive patients about their diseases and lifestyle advices has a significant impact on disease control and well-being

Factors associated with decision-making about end-of-life care by hemodialysis patients

The current cross sectional study is based on a questionnaire database on patients with end-stage renal disease (ESRD) to determine their preferences about end-of-life care and differences of certainty regarding the application of cardiopulmonary resuscitation and life sus-taining measures in case of cardiac arrest. The study was performed on 100 patients on hemo-dialysis for at least 2 years and not on the transplant list in two tertiary hospitals in Saudi Arabia; King Fahad National Guard in Riyadh and King Faisal Specialist Hospital in Jeddah in March 2007. More than two thirds of the surveyed patients were willing to make decisive decisions. Ha-ving more than 5 children was the only factor significantly associated with the ability to make de-cisive decisions; there was an insignificant association with factors such as marital status or non-Saudi nationality. Factors such as self-perception or disease curability, previous admissions to hospital or intensive care units, prior knowledge of mechanical ventilation, or cardiopulmonary resuscitation did not have any influence on making certain decisions on end-of-life care. There was a significant lack of knowledge in our study patients of cardiopulmonary resuscitation, me-chanical ventilation, and disease outcome

Advance care planning preferences among dialysis patients and factors influencing their decisions

To determine the resuscitation preferences of hemodialysis (HD) Saudi patients, we con-ducted a cross-sectional, observational descriptive questionnaire study in two major tertiary hospitals in Saudi Arabia from March to December 2007. We enrolled all the patients on HD for two years or more, and excluded the patients who were transplant candidates, confused, or demented. The questionnaire was com-posed of 4 sections. The first 3 sections were concerned with demographic data, education levels, employ-ment, family size, number of children, and functionality status besides knowledge about cardiopulmonary resuscitation (CPR), mechanical ventilation, and ICU admission. The fourth section contained different sce-narios and questions on personal and preferences such as end of life decisions, medical interventions, CPR, ICU admission, and the decision maker in these events. A total of 100 patients (53&#x0025; males, 67&#x0025; Saudis, and 85&#x0025; married) were enrolled in the study. The mean duration on dialysis was 6.0 years (&#x00B1; 4.1). More than 70&#x0025; of the patients viewed themselves as above average in the religiosity score, and 44&#x0025; disclosed a good life quality. More than 95&#x0025; had little or no knowledge about cardiac resuscitation, intubation, and mechanical ventilation. The majority of the patients authorized their treating physician to decide for them about cardiac resuscitation in case they did not make advanced directives and only 22&#x0025; believed that this decision should be made by their family members. If their physician believed their condition was hopeless, 77&#x0025; preferred to stay at home. We conclude that the majority of our patients had limited awareness about cardiac resuscitation measures. The majority of the patients trust their physicians to decide about the futility of resuscitation. Patients were able to decide reasonably well when they are well informed

Restless legs syndrome in patients on dialysis

Restless legs syndrome (RLS) is an extremely distressing problem experienced by patients on dialysis; the prevalence appears to be greater than in the general population, with a wide variation from 6.6&#x0025; to 80&#x0025;. The diagnosis of RLS is a clinical one, and its definition has been clarified and standardized by internationally recognized diagnostic criteria, published in 1995 by the International Restless Legs Syndrome Study Group (IRLSSG). This study was designed to find out the prevalence of RLS in Saudi patients with end-stage renal disease (ESRD) on maintenance dialysis. This is a cross sectional study carried out between May and Sept 2007 at two centers, King Abdulaziz Medical City-King Fahad National Guard Hospital (KAMC-KFNGH), Riyadh and King Faisal Specialist Hospital and Research Centre (KFHRC), Jeddah, Saudi Arabia. Data were gathered on 227 Saudi patients on chronic maintenance hemodialysis or chronic peritoneal dialysis. The prevalence of RLS was measured using IRLSSG&#x2032;s RLS Questionnaire (RLSQ). Potential risk factors for RLS including other sleep disorders, underlying cause of chronic renal failure, duration on dialysis, dialysis shift, biochemical tests and demographic data were also evaluated. The overall prevalence of RLS was 50.22&#x0025; including 53.7&#x0025; males and 46.3&#x0025; females. Their mean age was 55.7 &#x00B1; 17.2 years and mean duration on dialysis 40.4 &#x00B1; 37.8 months. Significant predictors of RLS were history of diabetes mellitus (DM), coffee intake, afternoon dialysis, gender and type of dialysis (P= 0.03, 0.01, &lt; 0.001, 0.05 and 0.009 respectively). Patients with RLS were found to be at increased risk of having insomnia and excessive daytime sleepiness (EDS) (P= &lt; 0.001 and 0.001, respectively). Our study suggests that RLS is a very common problem in dialysis population and was significantly associated with other sleep disorders, particularly insomnia, and EDS. Optimal care of dialysis patient should include particular attention to the diagnosis and management of sleep disorder

Antibody-mediated rejection and aHUS in renal graft recipient

A 46-year-old female who lost her native kidney function due a biopsy proven collapsing type of FSGS, had a living kidney transplant after which, she developed acute renal allograft dysfunction, thrombocytopenia and microangiopathichemolytic anemia five days post-transplant. Renal biopsy revealed acute antibody-mediated rejection (AMR). Genetic studies showed that this patient has a homozygous mutation of complement factor B (CFB) gene and heterozygous variant of C 3 gene consistent with atypical hemolytic uremic syndrome type 4. Intravenous immunoglobulin (IV IG) and plasma exchange did not resolve these abnormalities. Eculizumab and bortezomib, on other hand, were very effective

Medical grand rounds in Riyadh, Saudi Arabia: Current attitudes and barriers

Background: Medical grand rounds (MGRs) are considered key educational tools in most academic medical institutions. In this multi-center cross-sectional survey, we tried to determine the current attitudes of local medical practitioners to MGRs, as well as perceived barriers. Methodology: A total of 120 physicians from the National Guard Hospital, King Fahad Medical City, King Khalid University Hospital and King Faisal Specialist Hospital participated in the survey. The questionnaire consisted of statements on attitudes and perceived barriers against participating in MGRs, as well as participants′ levels of agreement. Results: Most participants attend MGRs regularly (94.2%), claiming that it is mandatory (88%). Participants also agreed that MGRs were important tools for continuing medical education (89.2%) and that they provided an opportunity to both present materials and interact with their colleagues in other divisions (86.7% and 81.6%, respectively). The vast majority of respondents agreed that "topic review/update" and "inviting guest speakers" were the two most preferred suggestions for improving MGRs (94.2% and 92.5%, respectively). Major barriers included constraints of time (43.3%) and topics that were not patient-related (40.8%). Conclusion: MGRs in the major Tertiary Hospitals in Riyadh are well attended, and the majority of the local practitioners believe in the positive effect of MGRs in delivering quality and up to date medical knowledge. Time and physician-specific issues were identified as major barriers that needed to be addressed in order to maximize participation of medical staff