Unlocking Applications of Cell-Free Biotechnology through Enhanced Shelf Life and Productivity of E. coli Extracts.

Cell-free protein synthesis (CFPS) is a platform biotechnology that enables a breadth of applications. However, field applications remain limited due to the poor shelf-stability of aqueous cell extracts required for CFPS. Lyophilization of E. coli extracts improves shelf life but remains insufficient for extended storage at room temperature. To address this limitation, we mapped the chemical space of ten low-cost additives with four distinct mechanisms of action in a combinatorial manner to identify formulations capable of stabilizing lyophilized cell extract. We report three key findings: (1) unique additive formulations that maintain full productivity of cell extracts stored at 4 °C and 23 °C; (2) additive formulations that enhance extract productivity by nearly 2-fold; (3) a machine learning algorithm that provides predictive capacity for the stabilizing effects of additive formulations that were not tested experimentally. These findings provide a simple and low-cost advance toward making CFPS field-ready and cost-competitive for biomanufacturing.

Manufacturing and quality assessment of allergenic extracts for immunotherapy: state of the art.

PURPOSE OF REVIEW: The recent developments in the manufacturing and quality assessment of allergenic extracts in Europe are summarized. RECENT FINDINGS: Quality assessment has always been a fundamental part of allergen product evaluation. New analytical methods have been reported that fill currently existing gaps in the characterization of commonly used allergen products. New types of products require innovative considerations and concepts for their assessment. Advanced standardization efforts aim at increasing reliability and comparability of analytical tools applied for allergen product characterization. In consequence, regulatory requirements are updated in line with such developments. SUMMARY: Current demands on the quality of allergen products ensure production of well characterized products of consistent quality. While experience with manufacturing processes and successful product characterization approaches increase, accompanying and continuous re-evaluation of underlying quality control and assessment concepts is being performed.

Human platelet lysate current standards and future developments.

A state-of-the-art workshop focused on the use of human platelet lysate (HPL) for cell therapy. The meeting established that HPL is used mainly as an adjunct material for ex vivo expansion of mesenchymal stem/progenitor cells (MSCs), where it is successfully used as a substitute for fetal bovine serum. HPL manufacturing as a cell expansion supplement is currently not yet uniformly standardized with regard to platelet source and production methodology. There are very few reports of HPL preparations manufactured specifically for direct clinical use. There exists an urgent need for controlled clinical studies for HPL and for standardization of product definition. Workshop participants also stated a need for consensus minimum release criteria to allow for better product definition and to limit variability in performance. The increasing use of cell-based therapies including MSCs has led to an increasing demand for HPL, either produced in blood establishments or large-scale manufacture by biopharmaceutical companies. The use of pooled donor platelets for HPL production may require the implementation of pathogen inactivation procedures and/or removal steps to improve the safety of advanced cell therapy products. There should also be a requirement for thorough risk assessments and risk mitigation steps, including the qualification of suppliers and identification of ingredients as well as meticulous monitoring of product quality and safety profiles. State-of-the-art regulatory approaches for HPL used for human cell propagation and PRP in direct clinical applications were reviewed.

Multiplex microbead measurements for the characterization of cat and ragweed allergen extracts.

BACKGROUND: Current assays for allergen extracts can measure either overall potency or the levels of individual allergens. OBJECTIVE: To develop a multiplex allergen extract potency assay (MAEPA) for allergen extracts that can concurrently measure individual allergens and characterize the overall allergen levels in the mixture. METHODS: Six anti-Fel d 1 and 6 anti-Amb a 1 recombinant antibodies were generated and were covalently bound to carboxy-labeled beads. Antibody-bound beads were then used to measure Fel d 1 and Amb a 1 levels in commercial cat hair and short ragweed pollen (SRP) extracts, respectively, using bead-based flow cytometry. These major allergen levels were compared with those obtained using a conventional antibody-based method. Allergen levels were calculated by comparing the half-maximal effective concentrations of dose-response curves analyzed using 4-parameter fits. Bead-antibody pairs were tested to determine whether the presence of additional bead-antibody pairs affected the apparent potency of the extract. RESULTS: Allergen contents of cat hair and SRP extracts determined using the MAEPA and anti-Fel d 1 and anti-Amb a 1 antibodies were comparable with potencies determined using conventional methods. Cross-interference from the concurrent use of multiple beads was minimal. Six lots of cat hair extract and 6 lots of SRP extract were tested. CONCLUSIONS: The MAEPA, a bead-based assay using recombinant antibodies, accurately determined Fel d 1 levels in cat hair allergenic extracts and Amb a 1 levels in SRP extracts. The results of this assay are reproducible and are consistent with data obtained using conventional methods.

Standardization of allergen extracts.

Allergens are molecules with the capacity to elicit IgE responses in humans. When stimulated with allergens, most allergic patients respond with production of IgE specific for several proteins/allergens in the source material. The standardization of allergen extracts is essential in order to control variability and to achieve consistency and reproducibility in a clinical setting. Because the IgE binding capacity of an allergen extract is related to the content of one or a few major allergens, it is important that the standardization procedure ensures consistency, not only in the overall IgE binding potency, but also in the content and ratio of individual major allergens. Owing to the complexity of allergen extracts, a key element in standardization of allergen extracts is the use of standards. This chapter describes the principles for standardization of allergen extracts to be used by research laboratories. Other chapters in this volume describe methods in detail.

Monoclonal IgE antibodies against birch pollen allergens: novel tools for biological characterization and standardization of allergens.

BACKGROUND: IgE antibodies are key players in immediate hypersensitivity reactions. Allergen characterization and standardization is usually based on the sera of allergic patients, whereas monoclonal IgE antibodies specific for clinically relevant allergens are very rare. OBJECTIVE: The aim of this study was to establish IgE mAbs specific for birch pollen allergens, because these are important inhalant allergens. METHODS: IgE-producing hybridomas were identified by using the highly sensitive rat basophilic leukemia cell mediator release assay with enhanced allergen stimulation by additional cross-linking with birch pollen-specific IgG antibodies. The obtained IgE mAbs were characterized by immunologic methods and by cDNA sequencing. RESULTS: Seven IgE mAbs specific for the birch pollen allergens Bet v 1 or Bet v 6 were obtained and were all biologically active in mast cell-based assays. Mediator release experiments with mAb combinations indicated that 2 different epitope regions were recognized on Bet v 1, whereas the 2 Bet v 6-specific mAbs bound to the same epitope region. After sensitization of rat basophilic leukemia cells with IgE mAbs, different amounts of Bet v 1 or Bet v 6 were detected in commercial diagnostic allergen reagents, whereas sensitization with polyclonal IgE resulted in similar allergenic potency of all products. CONCLUSIONS: IgE mAbs represent promising novel tools for allergen characterization and component-resolved standardization of allergen extracts.

Quality parameters for the production of mite extracts.

Mites present in house dust are of great etiological importance in type I hypersensitivity, with those belonging to the Dermatophagoides genus (D. pteronyssinus and D. farinae), of the Pyroglyphidae family, being the most frequent and principal source of allergens. For the production of allergenic extracts destined for specific diagnostic and treatment purposes of allergic diseases, the culture of such mites is absolutely necessary. In accordance with studies carried out in our laboratories to obtain adequate extracts, one must bear in mind the culture mite phase. Three growth phases have been distinguished for both species: latency phase (F1), growth phase (F2) in which the allergenic proteins are expressed with greater intensity, and death phase of the culture (F3). In the same study, the biological standardization of the extracts demonstrated that those produced from the maximum growth phase gave both in vitro and in vivo results, at least three times more sensitive than those from the other phases. We checked the reproducibility of the production method, obtaining different batches in similar conditions with a high homogeneity regarding allergenic activity. The sensitivity and specificity of the allergenic extracts depends just as much upon the production method as the standardization method. During the biological cycle of Dermatophagoides in culture, it is only from the maximum growth phase (F2), that allergenic extracts with an excellent diagnostic value, high sensitivity and specificity, can be obtained.

A collaborative study on the first international standard of Dermatophagoides pteronyssinus (house dust mite) extract.

A collaborative study was carried out to assess the suitability of a preparation to serve as the International Standard for Dermatophagoides pteronyssinus (house dust mite) extract. The proposed international standard of D. pteronyssinus, two additional freeze-dried extracts, and a commercially available skin testing solution were tested in the study. Nineteen laboratories in 11 different countries participated. The assay methods used included RAST inhibition, crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis, isoelectric focusing, quantitative skin testing, and various other methods for assessing total allergenic activity. In addition, six laboratories measured the quantity of antigen P1, and three laboratories measured antigen DpX in each of the preparations. On the basis of the results from this study, the World Health Organization established the preparation as the International Standard for D. pteronyssinus extract with an assigned unitage of 100,000 IU per ampule. The units refer both to the total allergenic activity of the ampule and to that of the individual allergens, such as P1 and DpX.

Standardization of house dust mite extracts. Liberation of allergenic and antigenic components in relation to extraction conditions.

The spontaneous release of house dust mite components from cultures of Dermatophagoides pteronyssinus into slightly buffered water was studied against time, using both continuous and discontinuous extraction procedures. It was shown that proteins, carbohydrates, IgE binding components and precipitating antigenic components were rapidly released from the house dust mite cultures, reaching a maximal liberation within 1 h of extraction. Repeated extractions of house dust mite cultures (discontinuous extraction) showed an additional release of IgE components but the IgE binding potency declined after successive extractions, while showing increasing release of immunological inactive components. IgE binding to antigens immobilized to polystyrene surfaces (IgE-ELISA) appeared to be less sensitive compared with cyanogen-bromide activated discs (IgE-RAST). It was concluded that extraction procedures of house dust mite cultures with short incubation time of 1 h or less are to be preferred.