Does the BioBLU 0.3f single-use scale to the BioFlo® 320 reuseable bioreactor on a matched volumetric oxygen mass transfer coefficient?

The volumetric oxygen mass transfer coefficient ([Formula: see text]) is an essential parameter in aerobic high-cell density fermentation where the availability of oxygen to growing microorganisms is a limiting factor. Bioprocess teams looking to scale-up/down between the Eppendorf BioBLU 0.3f single-use vessel and the BioFlo® 320 reusable vessel bioreactors may find it challenging using a matched [Formula: see text]. The maximum [Formula: see text] of the BioFlo® 320 reusable bioreactor was 109 h-1, which was approximately twice that of the BioBLU 0.3f single-use vessel. The results here show no overlap in [Formula: see text] values when both bioreactors were compared and thus conclude that scalability based on [Formula: see text] is not viable. The maximum [Formula: see text] of the Eppendorf BioBLU 0.3f single-use reported here was 47 h-1 compared to that of the manufacturer's value of 2500 h-1, indicating a 53-fold difference. This discrepancy was attributed to the incompatible sulfite addition method used by the manufacturer for estimation.

Kinetic modeling as a tool to understand the influence of cell culture process parameters on the glycation of monoclonal antibody biotherapeutics.

Glycation, the nonenzymatic reaction between the reducing sugar glucose and the primary amine residues on amino acid side chains, commonly occurs in the cell culture supernatant during production of therapeutic monoclonal antibodies (mAbs). While glycation has the potential to impact efficacy and pharmacokinetic properties for mAbs, the most common undesirable impact of glycation is on the distribution of charged species, often a release specification for commercial processes. Existing empirical approaches are usually insufficient to rationalize the effects of cell line and process changes on glycation. To address this gap, we developed a kinetic model for estimating mAb glycation levels during the cell culture process. The rate constant for glycation, including temperature and pH dependence, was estimated by fitting the kinetic model to time-course glycation data from bioreactors operated at different process settings that yielded a wide range of glycation values. The parameter values were further validated by independently estimating glycation rate constants using cell-free incubation studies at various temperatures. The model was applied to another mAb, by re-estimating the activation energy to account for effect of a glycation "hotspot". The model was further utilized to study the role of temperature shift as an approach to reduce glycation levels in the manufacturing process for mAb2. While a downshift in temperature resulted in lowering of glycation levels for mAb2, the model helped elucidate that this effect was caused due to contribution from changes in glucose consumption, mAb secretion and temperature, instead of a direct impact of temperature alone on the kinetic rate of glycation.

How does mild hypothermia affect monoclonal antibody glycosylation?

The application of mild hypothermic conditions to cell culture is a routine industrial practice used to improve recombinant protein production. However, a thorough understanding of the regulation of dynamic cellular processes at lower temperatures is necessary to enhance bioprocess design and optimization. In this study, we investigated the impact of mild hypothermia on protein glycosylation. Chinese hamster ovary (CHO) cells expressing a monoclonal antibody (mAb) were cultured at 36.5°C and with a temperature shift to 32°C during late exponential/early stationary phase. Experimental results showed higher cell viability with decreased metabolic rates. The specific antibody productivity increased by 25% at 32°C and was accompanied by a reduction in intracellular nucleotide sugar donor (NSD) concentrations and a decreased proportion of the more processed glycan structures on the mAb constant region. To better understand CHO cell metabolism at 32°C, flux balance analysis (FBA) was carried out and constrained with exometabolite data from stationary phase of cultures with or without a temperature shift. Estimated fluxomes suggested reduced fluxes of carbon species towards nucleotide and NSD synthesis and more energy was used for product formation. Expression of the glycosyltransferases that are responsible for N-linked glycan branching and elongation were significantly lower at 32°C. As a result of mild hypothermia, mAb glycosylation was shown to be affected by both NSD availability and glycosyltransferase expression. The combined experimental/FBA approach generated insight as to how product glycosylation can be impacted by changes in culture temperature. Better feeding strategies can be developed based on the understanding of the metabolic flux distribution.

The emergence of a learning healthcare system.

A learning healthcare system has emerged as a means of addressing the need to move evidence to the bedside in a manner that is meaningful and efficacious. The advent of electronic healthcare records has translated to massive quantities of clinical data; a learning healthcare system coupled with locally derived clinical rules will push practice-based evidence to the point of care where its application will mean improved quality and patient outcomes.

Return on investment imperative: the cost of care calculator for an evidence-based practice program.

The key elements of the evaluation process of an evidence-based practice (EBP) program and the infrastructure needed as identified by a large health care organization. The EBP evaluation program has 2 major elements for measuring success. The first component for evaluation is the impact on the clinical outcomes of care and the second is the fiscal implications of implementing the EBP. This article focuses on the fiscal evaluation component and describes a process to calculate the cost of care compared to before and after implementation of the EBP. The literature to support the care calculations is examined and a cost algorithm is described.

Intra individual variability in markers of proteinuria for normal subjects and those with cadmium induced renal dysfunction: interpretation of results from untimed, random urine samples.

The project aimed to help interpretation of urinary protein measurements, namely -2-microglobulin, retinol-binding protein, albumin and total protein in untimed, random urine samples as indicating significant changes in renal tubular reabsorption and glomerular permeability in an individual. A standard methodology used in clinical laboratory medicine was applied to calculate the intra-individual biological variation for these analytes. This parameter in conjunction with a laboratory's analytical variation allows definition of uncertainty about a single urine protein measurement, significant changes above normal variation in serial measurements within an individual and a defined level of maximum acceptable analytical imprecision. Repeat urine samples were obtained over a period of one week from a group of cadmium-exposed workers, 90% of whom had long-term tubular proteinuria, and a group of five unexposed volunteers with normal renal function. Dilute samples defined as having creatinines less than 3 mmol l-1 were excluded, as were urines with pH less than 5.5 for -2-microglobulin. Samples were analysed twice after randomisation in large batches. There was no evidence of any diurnal variation in the four protein measurements from samples collected between early morning and 16:00 hours. Creatinine or specific gravity correction of urine results for all four proteins only marginally reduced the uncertainty associated with an individual measurement asreflecting the true excretion value. For those subjects with defined tubular proteinuria, variability in retinol-binding protein excretion was less than that for -2- microglobulin. About 30% of the samples had urine pHs of 5.5 or less where -2- microglobulin degradation occurs. Using our laboratory analytical precision the minimum changes between serial creatinine-corrected measurements that are needed to be considered statistically significant (p < 0.05) is 110% for retinol-binding protein, 177% for -2-microglobulin, 70% for total protein, and 81% for albumin. Unlike published data for cadmium and mercury, the use of creatinine or specific gravity correction of random untimed urine samples for the urinary proteins does not make a large improvement to the interpretation of data by reducing the uncertainty associated with a measurement. There are significant advantages to the use of retinol-binding protein in contrast to -2- microglobulin as an indicator of renal tubular damage. Levels for defined acceptable analytical precision are calculated for laboratories undertaking protein estimations. The data in this report will help in the interpretation of urinary protein measurements without monitoring cadmium workers.