Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: Improving the efficiency of cell line generation

Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide‐ranging transfectant population. This identification process is on the critical path for first‐in‐human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One‐hundred and seventy‐five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

An improved method for the detection and quantification of recombinant protein in transgenic plants

A sensitive method has been developed for the detection of recombinant protein produced as a result of gene transfer into plants. This method is based upon antibody binding, which is then visualized using enhanced chemiluminescence and recorded on x-ray film for long-term storage. The technique is simple, rapid and reliable and can be used to screen large numbers of transgenic plants. Several plant species have been successfully tested in this way for a range of recombinant proteins.     

A gas chromatographic method for quantification of detergents frequently used in membrane protein structural studies

A novel and reliable gas chromatography-flame ionization detection (GC-FID) method that can separate and quantify detergents frequently used in membrane protein structural studies has been developed. Different detergents were identified through FID peaks with different retention times. A quadratic regression curve was found to fit the integrated FID peak area against different detergent concentrations. Detergents can be quantified as low as the nanogram level: lauryl-dimethylamine-N-oxide (LDAO), 5 ng; dodecyl maltoside (DDM), 10 ng; and dodecyl phosphocholine (DPC), 50 ng. This method can be applied directly to measure detergent concentration and molar ratio of membrane protein to detergents during membrane protein extraction, purification, concentration, and crystallization.     

A double epitope tag for quantification of recombinant protein using fluorescence resonance energy transfer

The expression of recombinant proteins is a well-accepted technology, but their detection and purification often require time-consuming and complicated processes. This paper describes the development of a novel double epitope tag (GEPGDDGPSGAEGPPGPQG) for rapid and accurate quantification of recombinant protein by a homogeneous immunoassay based on fluorescence resonance energy transfer. In our double epitope tagging system, recombinant proteins can be simply measured on a microtiter plate by addition of a pair of fluorophore-labeled monoclonal antibodies (their epitopes; GEPGDDGPS and GPPGPQG). The sensitivity of the immunoassay with an incubation time of only 5 min is almost equal to that of labor-intensive Western blotting. In addition, culture media and extracts of host cells generally used for protein expression have little effect on this immunoassay. To investigate the utility of our proposed tag for protein production, several different proteins containing this tag were practically expressed and purified. The data presented demonstrate that the double epitope tag is a reliable tool that can alleviate the laborious and troublesome processes of protein production.     

Emerging trends in plasma-free manufacturing of recombinant protein therapeutics expressed in mammalian cells

Mammalian cells are the expression system of choice for therapeutic proteins, especially those requiring complex post-translational modifications. Traditionally, these cells are grown in medium supplemented with serum and other animal- or human-derived components to support viability and productivity. Such proteins are also typically added as excipients and stabilizers in the final drug formulation. However, the transmission of hepatitis B in the 1970s and of hepatitis C and HIV in the 1980s through plasma-derived factor VIII concentrates had catastrophic consequences for hemophilia patients. Thus, due to regulatory concerns about the inherent potential for transmission of infectious agents as well as the heterogeneity and lack of reliability of the serum supply, a trend has emerged to eliminate the use of plasma-derived additives in the production and formulation of recombinant protein therapeutics. This practice began with products used in the treatment of hemophilia and is progressively expanding throughout the entire industry. The plasma-free method of producing recombinant therapeutics is accomplished by the use of both cell culture media and final product formulations that do not contain animal- or human-derived additives. A number of recombinant therapeutic proteins for the treatment of several different diseases have been produced by plasma-free processes, with the objective of improving safety by eliminating blood-borne pathogens or by reducing immunogenicity. This review describes the factors that drove the development of plasma-free protein therapeutics and provides examples of advances in manufacturing that have made possible the removal of human and animal-derived products from all steps of recombinant protein production.     

Assessing the impacts of biochar-blended urea on nitrogen use efficiency and soil retention in wheat production

Improving nitrogen (N) use efficiency (NUE) in crop plants is important to reduce the negative impact of excessive N on the environment. Although biochar-blended fertilizer had been increasingly tested in crop production, the fate of fertilized N in soil and plant had not been elucidated in field conditions. In this study, a novel biochar-blended urea (BU) was prepared by pelleting maize straw biochar, bentonite, sepiolite, carboxymethylcellulose sodium, and chitosan with urea (commercial urea without biochar [CU]). N fertilization in a winter wheat field was treated with BU and CU at both 265 kg N ha^?1 (HL) and 186 kg N ha^?1 (LN). Within a treatment plot, a microplot was fertilized with ¹⁵N-labeled urea at a relevant N level. We investigated the influence of fertilizer management on biomass, grain yield, bioaccumulation of nutrient, soil properties, ¹⁵N isotopic abundance, and greenhouse gas emissions. Microscopic and spectroscopic analysis showed that micro/nanonetwork of biochar could bind N to form a loss control agglomerated particle, and organo-mineral coatings on BU may protect N from quick release. Compared with CU, BU significantly increased grain yield by 13% and 38%, and grain N allocation by 19% and 55%, respectively, at HN and LN level. The total recovery of urea ¹⁵N in wheat plant (¹⁵N based NUE) was 32.8% under CU regardless of N rates but increased to 41.7% (HN rate) and 56.3% (LN rate) under BU. Whereas, the soil proportion (soil residual ¹⁵N) was 20.1% and 13.4% under CU but 32.5% and 18.8% under BU, in 0-20cm topsoil, respectively, at HN and LN rate. Compared with the CU, BU had no effect on CO₂ and CH₄ emissions but significantly reduced the total N₂O emission by 23%–28%. These important findings suggested that BU can be beneficial to uplift plant NUE to reduce reactive N loading but boost crop production.     

Process simulation for recombinant protein production: cost estimation and sensitivity analysis for heparinase I expressed in Escherichia coli

Heparinase I from flavobacterium heparinum has several potential clinical applications; the resulting high demands on protein purity and quantity can be met by recombinant expression in Escherichia coli. Based on laboratory scale experiments with insoluble heparinase I expression followed by renaturation, a process for production of 3 kg/year of heparinase I was designed. We present a comparative analysis of the production costs of soluble and insoluble heparinase I expression, as well as a generalized approach to sensitivity analysis, based on perturbation around a base case design scenario. This may assist focusing further development on process steps for which improvements both are feasible and result in significant cost saving. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 575-582, 1997.     

A two-dimensional thin-layer chromatographic procedure for the estimation of plasmalogens

1. The use of two-dimensional thin-layer chromatography is described that allows the rapid and simultaneous determination of phospholipid classes and their constituent plasmalogens. 2. The method is based on the specific hydrolysis of plasmalogens to (2-acyl) lysophospholipid in the presence of a mercuric chloride spray reagent. 3. The proportion of mercuric chloride-labile phospholipid present in each phospholipid class, calculated on the basis of phosphorus recoveries from the charred chromatogram, was compared with the proportion of long-chain aldehyde and of total lipid phosphorus found in small-scale preparations of each class of phospholipid. 4. The method permits the determination of individual plasmalogens on preparations containing as little as 0.2mug.atom of total lipid phosphorus.     

The use of cyanate for the determination of NH 2 -terminal residues in N-acylated proteins

The results show that when the cyanate method is applied to proteins with certain N-acyl substituents, the terminal residues in their acylated form can appear in the hydantoin fraction and give rise to misleading conclusions on homogeneity. The survival of the N-acyl amino acid, and the loss of hydantoin, are governed by the conditions employed in the cyclization step. The blocked termini in N-acylated proteins can be demonstrated by using modified cyclization conditions.     

High-performance liquid chromatographic method for the quantification of unbound evernimicin in human plasma ultrafiltrate

A rapid HPLC method was developed for quantification of unbound evernimicin in human plasma. Protein-free samples prepared by ultrafiltration were injected directly onto a polymeric reversed-phase column and the eluent monitored at 302 nm. Evernimicin that eluted within 3.5 min was well resolved from endogenous components. Linearity was established between peak height and evernimicin concentration from 25 to 2500 ng/ml. Assay precision (C.V.) was within 5% while bias was no greater than 3%. This method has been used for the ex vivo assessment of evernimicin protein binding in human plasma from safety and tolerance as well as liver dysfunction and renal insufficiency studies.     

The use of whole barley diets fortified with solutions of urea,minerals and vitamins for lambs

For one experiment 45 early-weaned lambs were given one of the following five diets from weaning to slaughter: (1) whole barley with urea, minerals and vitamins added as a concentrated solution; (2) as diet (1) plus 4 g/kg of sodium sulphate in solution; (3) as diet (2) plus 1.2 g of methionine-hydroxyanalogue (MHA)/kg; (4) as diet (2) plus 2.5 ml of 40% formaldehyde added per kg; (5) a control diet containing whole barley and 100 g/kg of a pelleted supplement based on fish meal. Growth rates (g/d) for the five treatments were 218, 253, 253, 256 and 292. Addition of sulphate significantly increased growth rate and food utilization while MHA had no effect; formalin treatment reduced digestibility and food utilization.In a second experiment 58 lambs were used to study the effect of protein supplements for lambs weaned at various ages and weights. Diets similar to (2) and (5) from Expt. 1 were used, while an intermediate diet (6) was made from an equal mixture of diets (2) and (5). As weaning age increased and as live weight at weaning increased, the difference in growth rate and food utilization between lambs receiving diet (2) and those receiving diets (5) and (6) decreased.It is suggested that for most sheep production systems in which concentrates are used either as the sole feed or as supplements, simple fortification of whole grain with the necessary nutrients is all that is required to achieve optimum results.