Viral inactivation for pH-sensitive antibody formats such as multi-specific antibodies.

Recently developed multi-specific antibody formats enable new therapeutic concepts. Conveniently, formats with an Fc domain allow purification in well-established mAb platform processes. However, due to the structural complexity of the formats, the assembled molecules may be sensitive to extreme pH commonly used for viral inactivation. An alternative to low pH incubation for virus inactivation is the use of a mixture of tri-n-butyl phosphate (TnBP, solvent) and Polysorbate 80 (PS80, detergent). While TnBP is toxic, this combination has a long history of use in the manufacturing of human plasma-derived products that are sensitive to low or high pH incubation. Data are provided demonstrating that the solvent/detergent (S/D) treatment using TnBP and PS80 can be successfully used for pH-sensitive, multi-specific antibody formats in the clarified cell culture fluid (CCCF). A different placement of the S/D within the purification process, namely during the capture by Protein A (PA), has been evaluated. This alternative placement allows effective viral inactivation by S/D while preserving the viral reduction and viral inactivation achieved through the PA step itself, enabling the cumulation of these effects. Furthermore, the process alternative simplifies the liquid handling by reducing the added volumes of the required S/D liquids, thus reducing the amount of toxic TnBP to a minimum. Data are shown demonstrating a complete removal of TnBP and PS80 in the process.

Enhanced Downstream Processing for a Cell-Based Avian Influenza (H5N1) Vaccine.

H5N1 highly pathogenic avian influenza virus (HPAIV) infections pose a significant threat to human health, with a mortality rate of around 50%. Limited global approval of H5N1 HPAIV vaccines, excluding China, prompted the need to address safety concerns related to MDCK cell tumorigenicity. Our objective was to improve vaccine safety by minimizing residual DNA and host cell protein (HCP). We developed a downstream processing method for the cell-based H5N1 HPAIV vaccine, employing CaptoTM Core 700, a multimodal resin, for polishing. Hydrophobic-interaction chromatography (HIC) with polypropylene glycol as a functional group facilitated the reversible binding of virus particles for capture. Following the two-step chromatographic process, virus recovery reached 68.16%. Additionally, HCP and DNA levels were reduced to 2112.60 ng/mL and 6.4 ng/mL, respectively. Western blot, high-performance liquid chromatography (HPLC), and transmission electron microscopy (TEM) confirmed the presence of the required antigen with a spherical shape and appropriate particle size. Overall, our presented two-step downstream process demonstrates potential as an efficient and cost-effective platform technology for cell-based influenza (H5N1 HPAIV) vaccines.

Staged purification of phosphogypsum using pH-dependent separation process.

Phosphogypsum (PG) is an industrial by-product of the transformation of phosphate rocks. For decades, PG has been a source of environmental concern due to the massive amount produced thus far, i.e., 7 billion tons, with a current production rate of 200-280 million tons per year. Phosphate minerals contain various impurities that precipitate and concentrate within PG. These impurities hinder PG usability in various sectors. This paper aims to purify PG using an innovative process based on staged valorization of PG. Initially, PG dissociation by ethylenediaminetetraacetic acid (EDTA) was optimized. After screening of different parameters and monitoring the ionic conductivity of solutions, it was disclosed that a pH-dependent solubilization process in the presence of EDTA resulted in high solubility of PG, up to 11.82 g/100 mL at pH > 11. Subsequently, a recovery of the purified PG by selective precipitation of calcium sulfate dihydrate (CSD) from obtained filtrate through pH adjustment to 3.5 were investigated. An abatement of 99.34% Cr, 97.15% Cd, 95.73% P2O5, 92.75% Cu, 92.38% Al2O3, 91.16% Ni, 74.58% Zn, 72.75% F, 61.43% MgO, 58.8% Fe2O3, 56.97% K2O, and 55.41% Ba was achieved. The process relied on the variation of EDTA chelation properties towards monovalent, divalent, and trivalent cations at different pHs. According to the findings of this study, a staged purification process in the presence of EDTA is an effective method for removing impurities from the industrial PG.

Optimized Separation of Carthamin from Safflower by Macroporous Adsorption Resins and Its Protective Effects on PC12 Cells Injured by OGD/R via Nrf2 Signaling Pathway.

The growing demand for safe natural products has reignited people's interest in natural food pigments. Here, we proposed the use of macroporous adsorption resins (MARs) to separate and purify carthamin from safflower. The optimal parameters for carthamin purification with HPD400 MAR were determined as follows: a mass ratio of crude carthamin in sample solution to wet resin of 0.3, a crude carthamin solution concentration of 0.125 g·mL-1, a pH of 6.00, a sample volume flow rate of 0.5 mL·min-1, an ethanol volume fraction of 58%, an elution volume of 4 BV, and an elution volume flow rate of 1.0 mL·min-1. Under the above purification conditions, the recovery rate of carthamin was above 96%. Carthamin dramatically improved the survival rate of PC12 cells damaged by oxygen-glucose deprivation/reoxygenation and protected them from oxidative stress by inhibiting the generation of reactive oxygen species and increasing the total antioxidant capacity and glutathione (GSH) levels. Carthamin promoted extracellularly regulated protein kinase phosphorylation into the nucleus, permitting Nrf2 nuclear translocation and upregulating the gene expression of the rate-limiting enzymes glutamate-cysteine ligase catalytic subunit and glutamate-cysteine ligase regulatory subunit of GSH synthesis to obliterate free radicals and exert antioxidant effects. This study revealed the purification method of carthamin and its antioxidant protective effects, providing important insights into the application of carthamin in functional foods.

Monoclonal Antibody Sequence Variants Disguised as Fragments: Identification, Characterization, and Their Removal by Purification Process Optimization.

During early stage development of a therapeutic IgG1 monoclonal antibody, high levels of low molecular weight (LMW) peaks were observed by high performance size-exclusion chromatography and capillary electrophoresis. Further characterization of the LMW peak enriched HPSEC fractions using reversed phase liquid chromatography coupled to mass spectrometry showed these LMW species were 47 kDa and 50 kDa in size. However, the measured masses could not be matched to any fragments resulting from peptide bond hydrolysis. To identify these unknown LMW species, molecular characterization methods were employed, including high-throughput sequencing of RNA. Transcriptomic analysis revealed the LMW species were generated by mis-splicing events in the heavy chain transcript, which produced truncated heavy chain products that assembled with the light chain to mimic the appearance of fragments identified by routine purity assays. In an effort to improve product quality, an optimized purification process was developed. Characterization of the process intermediates confirmed removal of both LMW species by the optimized process. Our study demonstrates that deep-dive analytical characterization of biotherapeutics is critical to ensure product quality and inform process development. Transcriptomic analysis tools can help identify the cause of unknown species, and plays a key role in product and process characterization.

Extractive fermentation for process integration of protease production by Aspergillus tamarii Kita UCP1279 and purification by PEG-Citrate Aqueous Two-Phase System.

The present study evaluated the influence of the variables polyethylene glycol (PEG) molar mass, pH, PEG concentration and sodium citrate concentration in the integrated production of the protease from Aspergillus tamarii Kita UCP1279 by extractive fermentation, obtaining as a response the partition coefficient (K), activity yield (Y) and concentration factor (CF). The enzyme preferably partitioned to the top phase and obtained in the system formed by variables MPEG = 400 g mol-1, CPEG = 20% (w w-1), and CCIT = 20% (w w-1) and pH 6, in this condition were obtained CF = 1.90 and Y = 79.90%. The protease showed stability at a temperature of 60 °C for 180 min, with optimum temperature 40 °C and pH 8.0. For the ions and inhibitors effects, the protease activity increased when exposed to Fe2+, Ca2+ and Zn2 + and inhibited by EDTA, being classified as metalloprotease. The kinetic parameters Km (35.63 mg mL-1) and Vmax (1.205 mg mL-1 min-1) were also estimated. Thus, the protease showed desirable characteristics that enable future industrial applications, especially, for beer industry.

A flow-through chromatography purification process for Vero cell-derived influenza virus (H7N9).

Immunization is the most effective way to respond to an influenza epidemic. To produce Vero cell-derived influenza vaccines, a more efficient, stable and economical purification process is required. In this study, we purified the H7N9 influenza virus grown in Vero cells that were cultured in a serum-free medium by using a combination of anion exchange chromatography (AEC) and ligand-activated core chromatography (LCC), which avoids the virus capture step. After purification, 99.95 % host cell DNA (hcDNA) (final concentration: 28.69 pg/dose) and 98.87 % host cell protein (HCP) (final concentration: 28.28 ng/dose) were removed. The albumin content was 11.36 ng/dose. All these remnants met the current Chinese Pharmacopoeia and WHO requirements. The final virus recovery rate was 58.74 %, with the concentration of hemagglutinin recorded at 132.12 µg/mL. The flow-through chromatography purification process represents an alternative to the existing processes for cell-derived influenza viruses and might be suitable for the purification of other viruses as well.

Optimization of Extraction and Purification Process of Total Iridoids and Total Crocins in Gardeniae Fructus / 中国实验方剂学杂志

ObjectiveTo optimize the extraction and purification process of Gardeniae Fructus for industrial production， and to obtain the total iridoid and total crocin extracts. MethodOrthogonal test was used to optimize the water extraction process by taking contents of geniposide， genipin gentiobioside， gardenoside， crocin-1 and crocin-2 as indicators and the decocting time， decocting times and water amount as factors. The purification process was optimized by single factor test， and four different types of macroporous adsorption resins were screened. The process conditions such as resin type， maximum loading amount， water washing amount， ethanol concentration， ethanol dosage， and flow rate of sample loading were mainly investigated. In addition， the drying methods （vacuum drying and spray drying） of the extract were investigated， and a pilot scale-up verification test was carried out. ResultThe optimal water extraction process of Gardeniae Fructus was to add 15， 10 times the amount of water for decocting twice， 1 h each time. The optimal purification process was as follows：the water extract through SP825L macroporous resin column， the amount of crude drug-the amount of resin （1∶1.5）， the sample loading flow rate of 3 BV h-1， adding 2 BV of water to remove impurities， adding 4 BV of 30% ethanol to obtain the iridoid part， then adding 3 BV of 70% ethanol to obtain the crocin part， collecting the ethanol lotion， and drying at 70 ℃. Under these conditions， the extraction amount of total iridoids was 590.75 mg·g-1 with the transfer rate of 70.48%， and the yield of dry extract was 8.89%. The extraction amount of total crocins was 83.37 mg·g-1 with the transfer rate of 22.20%， and the dry extract yield was 2.60%. ConclusionThe optimized extraction and purification process is stable and feasible with high extraction rate of active components， which is suitable for the industrial extraction and purification of active parts of Gardeniae Fructus.

Measurement of High Protein Concentrations by Optical Rotation: A Case Study for Monitoring of Monoclonal Antibody Drug Downstream Processes.

BACKGROUND: Recent advancements in cell engineering and bioreactor engineering have enabled high monoclonal antibody (mAb) concentrations in harvested solutions for the downstream process (DSP).

Methods: As many unit operations such as capture chromatography, polish chromatography, membrane filtration, virus inactivation, virus filtration, and concentration by ultrafiltration are involved in DSP, it is crucial to monitor the process carefully in order to perform reliable and stable DSP operations. One of the most important signals (process parameter) to be monitored is the protein concentration CP. Although various methods are available, most of them are not suited for measuring high CP. In this paper, we have developed a method for measuring very high CP by optical rotation (OR).

Result: Linear correlations were confirmed between OR and Cp in the range CP = 0 to 80 g/L for mAbs with high repeatability and small variation coefficients. This method was applied to the monitoring of CP in the opaque (colored) solution during the cell culture. The CP by OR was in good agreement with those by the standard Protein A HPLC method.

Conclusion: Monitoring of high CP by OR is expected to be an efficient process analytical tool (PAT) for DSP.

A simple 2-step purification process of α-amylase from Bacillus subtilis: Optimization by response surface methodology.

Purification of extracellular α-amylase from Bacillus subtilis was carried out via fractional precipitation by acetone and ion exchange chromatography. These steps provide fast precipitation as well as purification of α-amylase to improve enzyme purity, activity and stability. Compared with two-phase methods in which the yield was less than 1, this method resulted in a yield of more than 3. Moreover, 95% of acetone was recovered that enhanced the economy of the downstream process. Using the data provided by 2D electrophoresis, purification was done by a single step ion exchange chromatography. The enzyme exhibited a molecular mass (SDS-PAGE) of 50KD and the pI of 5. Maximum "yield" and "purification fold" were achieved through optimization of operation parameters such as volume and flowrate of loaded protein using response surface methodology (RSM). 0.5ml of loaded protein at a flow rate of 0.5 ml/min was purified as 48 folds and achieved a specific activity of 524 U/mg.

Critical Assessment of Purification and Analytical Technologies for Enveloped Viral Vector and Vaccine Processing and Their Current Limitations in Resolving Co-Expressed Extracellular Vesicles.

Viral vectors and viral vaccines are invaluable tools in prevention and treatment of diseases. Many infectious diseases are controlled using vaccines designed from subunits or whole viral structures, whereas other genetic diseases and cancers are being treated by viruses used as vehicles for delivering genetic material in gene therapy or as therapeutic agents in virotherapy protocols. Viral vectors and vaccines are produced in different platforms, from traditional embryonated chicken eggs to more advanced cell cultures. All these expression systems, like most cells and cellular tissues, are known to spontaneously release extracellular vesicles (EVs). EVs share similar sizes, biophysical characteristics and even biogenesis pathways with enveloped viruses, which are currently used as key ingredients in a number of viral vectors and licensed vaccine products. Herein, we review distinctive features and similarities between EVs and enveloped viruses as we revisit the downstream processing steps and analytical technologies currently implemented to produce and document viral vector and vaccine products. Within a context of well-established viral vector and vaccine safety profiles, this review provides insights on the likely presence of EVs in the final formulation of enveloped virus products and discusses the potential to further resolve and document these components.

Flow management strategies for a connected purification process.

This paper describes different flow management strategies for a connected purification process which includes two polishing steps, virus filtration and tangential flow filtration. Connecting these unit operations avoids introducing large intermediate product pool vessels in small manufacturing facilities. However, a connected-downstream process requires an elaborate control strategy enabling multiple unit operations to function as a single unit. The key strategy to enable the connected-downstream process is a robust management of flow disparities among unit operations. During a typical ultrafiltration step, product concentration increases as mass is added to the retentate tank, leading to a permeate flux decline. In a connected-downstream process, the inlet stream is directly connected to the prior unit operation and any decrease in permeate flow rate could cause a flow disparity. Four different flow management approaches are proposed to manage potential flow disparities and their advantages and challenges are discussed. Bench-scale results of these strategies are presented and evaluated.

In-Line Monitoring of Downstream Purification Processes for VSV Based SARS-CoV-2 Vaccine Using a Novel Technique.

The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) increases the need for a rapid development of efficient vaccines. Among other vaccines in clinical trials, a recombinant VSV-∆G-spike vaccine was developed by the Israel Institute for Biological Research (IIBR) and is being evaluated. The development of an efficient downstream purification process (DSP) enables the vaccine to be advanced to clinical trials. The DSP must eliminate impurities, either process- or product-related, to yield a sufficient product with high purity, potency and quality. To acquire critical information on process restrictions and qualities, the application of in-line monitoring is vital and should significantly impact the process yield, product quality and economy of the entire process. Here, we describe an in-line monitoring technique that was applied in the DSP of the VSV-∆G-spike vaccine. The technique is based on determining the concentrations of metabolites, nutrients and a host cell protein using the automatic chemistry analyzer, Cobas Integra 400 Plus. The analysis revealed critical information on process parameters and significantly impacted purification processes. The technique is rapid, easy and efficient. Adopting this technique during the purification process improves the process yield and the product quality and enhances the economy of the entire downstream process for biotechnology and bio pharmaceutical products.

Degradation of common polymers in sewage sludge purification process developed for microplastic analysis.

To enable and/or facilitate analysis of microplastics from environmental samples, a purification process is required to reduce the organic matter content. The development of such process has as one main concern, besides achieving efficient organic matter reduction, the preservation of the microplastics. In this study, a three-step method for sewage sludge purification was proposed employing sodium dodecyl sulfate and hydrogen peroxide. The effects of the purification method on seven polymers (LLDPE, HDPE, PP, PS, PET, PA66 and SBR) were evaluated in terms of mass change, surface characteristics, mechanical properties, thermal properties and functional groups change. It was also assessed how the polymers were affected by the purification chemicals without the presence of sewage sludge. The purification process led to changes in all tested plastics, but in different intensities. LLDPE, HDPE, PP, PS and PET did not suffer considerable degradation. PET was more affected by hydrolysis than oxidation. On the other hand, the integrities of PA66 and SBR were noticeably affected. The effects of the purification process were considered to be due to the plasticizer behavior of water and oxidation on PA66 and loss of filler and oxidation on SBR. For both polymers there was a reduction on the tensile strength of around 50-60% after the purification, indicating they could be prone to fragmentate into smaller pieces along the process. After purification, PA66 also started to decompose at a temperature around 10 °C lower comparing to virgin samples. Except for SBR, the presence of sewage sludge and its oxidation was more harmful to the polymers than the purification chemicals without the presence of sewage sludge. This study serves as an evaluation of the effects of the purification process on the degradation of microplastics and a methodology for such assessment when designing a purification process.

Occurrence of antibiotics in rural drinking water and related human health risk assessment.

Antibiotic residues in drinking water can have a negative impact on both human and environmental health. However, drinking water purification processes employed in rural areas are often less complicated than those used in urban areas. The occurrence of antibiotic residues in rural drinking water and their potential effects on residents' health remains to be established. In this study, we measured antibiotic levels in rural drinking water using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), and evaluated the associated health risks based on Chinese population exposure parameters. Twenty-three antibiotics were detected in drinking water samples, of which fluoroquinolones and macrolides were the most common. The type and concentration of antibiotics in drinking water were affected both by the quality of the water source and by the water purification process used. The health risks associated with antibiotics in drinking water were within acceptable levels and likely to have little impact on human health. Of the antibiotics detected, salinomycin presented the greatest risk to human health. These findings can help to play a role in devising strategies to ensure drinking water safety.

Optimization of total polyphenol purification from purple tea by macroporous resin / 药学实践杂志

Objective To screen the macroporous adsorption resin suitable for the separation and purification of total polyphenols from purple tea and establish the purification process parameters to prepare high-purity total polyphenols from purple tea. Methods The static adsorption-elution test was used to screen macroporous adsorption resin for the purification of total polyphenols from purple tea. Based on the single factor test, the comprehensive score of adsorption rate was used as the index to investigate the effects of different factors on the purification process and identify the optimal parameters for the purification process. Those factors included sample concentration, the pH value of the sample solution, the ratio of column diameter to height, sample size, ethanol percentage in the eluent, eluent volume and elution flow rate. Results The best process parameters for purification of total polyphenols from purple tea by AB-8 macroporous adsorption resin were as following. The sample concentration was 375 μg/ml with flow rate 2 ml/min. The sample volume was 3 BV. The sample solution pH was 2. The ratio of colume diameter to height was 1∶6. The impurities were removed first by water 3 BV. 50% ethanol 4 BV was used for elution with flow rate 2 ml/min. Conclusion AB-8 macroporous resin was selected for the purification of polyphenols from purple tea under the optimized technological conditions. The mass fraction of total polyphenols increased from 40.2% to an average of 69.8%. The solid content decreased from 56.0 mg to 29.9 mg. The established purification process has good stability and feasibility. It can be used as a purification process for total polyphenols from purple tea.

Laboratory-Scale Lentiviral Vector Production and Purification for Enhanced Ex Vivo and In Vivo Genetic Engineering.

Lentiviral vectors (LVs) are increasingly employed in gene and cell therapy. Standard laboratory production of LVs is not easily scalable, and research-grade LVs often contain contaminants that can interfere with downstream applications. Moreover, purified LV production pipelines have been developed mainly for costly, large-scale, clinical-grade settings. Therefore, a standardized and cost-effective process is still needed to obtain efficient, reproducible, and properly executed experimental studies and preclinical development of ex vivo and in vivo gene therapies, as high infectivity and limited adverse reactions are important factors potentially influencing experimental outcomes also in preclinical settings. We describe here an optimized laboratory-scale workflow whereby an LV-containing supernatant is purified and concentrated by sequential chromatographic steps, obtaining biologically active LVs with an infectious titer and specific activity in the order of 109 transducing unit (TU)/mL and 5 × 104 TU/ng of HIV Gag p24, respectively. The purification workflow removes >99% of the starting plasmid, DNA, and protein impurities, resulting in higher gene transfer and editing efficiency in severe combined immunodeficiency (SCID)-repopulating hematopoietic stem and progenitor cells (HSPCs) ex vivo, as well as reduced activation of inflammatory responses ex vivo and in vivo as compared to TU-matched, laboratory-grade vectors. Our results highlight the value of accessible purified LV production for experimental studies and preclinical testing.

Extraction and structural analysis of Angelica sinensis polysaccharide with low molecular weight and its lipid-lowering effect on nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is one of the prevalent and typical chronic liver diseases. In this study, we extracted a novel Angelica sinensis polysaccharide (ASP) with low molecular weight (MW) of 3.2 kDa through optimized "one-step" purification process. The major monosaccharide components of ASP were mannose, rhamnose, glucuronic acid, galactose, arabinose, and xylose with weight ratio of 0.23:0.17:14.41:0.39:1.68:0.87, respectively. Herein, "small" ASP could serve as an effective therapeutic option for NAFLD both in free fatty acid-induced L02 models and in high-fat diet-induced mice models. Results revealed that low MW ASP dose-dependently decreased TG, TC in vitro and TG, TC, ALT, HDL-C, and LDL-C in vivo. Oil Red O-positive area and Nile red fluorescence intensity decreased in ASP treatment groups both in vitro and in vivo which suggested ASP could reduce lipid accumulation and fatty regeneration. Hematoxylin-eosin staining results shown a decrease in hepatocytes ballooning indicating that ASP could ameliorate liver lipid degeneration. Briefly, a novel polysaccharide with low MW was successfully obtained which can prospectively act as NAFLD therapy.

Continuous Monitoring of Air Purification: A Study on Volatile Organic Compounds in a Gas Cell.

Air pollution is one of the major environmental issues that humanity is facing. Considering Indoor Air Quality (IAQ), Volatile Organic Compounds (VOCs) are among the most harmful gases that need to be detected, but also need to be eliminated using air purification technologies. In this work, we tackle both problems simultaneously by introducing an experimental setup enabling continuous measurement of the VOCs by online absorption spectroscopy using a MEMS-based Fourier Transform infrared (FTIR) spectrometer, while those VOCs are continuously eliminated by continuous adsorption and photocatalysis, using zinc oxide nanowires (ZnO-NWs). The proposed setup enabled a preliminary study of the mechanisms involved in the purification process of acetone and toluene, taken as two different VOCs, also typical of those that can be found in tobacco smoke. Our experiments revealed very different behaviors for those two gases. An elimination ratio of 63% in 3 h was achieved for toluene, while it was only 14% for acetone under same conditions. Adsorption to the nanowires appears as the dominant mechanism for the acetone, while photocatalysis is dominant in case of the toluene.

Response-surface methodology for the production and the purification of a new H2 O2 -tolerant alkaline protease from Bacillus invictae AH1 strain.

This work deals with the optimization of the culture conditions of Bacillus invictae AH1 in order to increase the production level of the proteolytic activity. Response-surface methodology (RSM) was applied for the most significant fermentation parameters (concentration of wheat bran and K2 HPO4 /KH2 PO4 ) that were earlier identified by Plackett-Burman Design from seven possible factors. A central composite design was used and the quadratic regression model of producing active protease was built. A maximum protease activity was reached and validated experimentally, using a maximum wheat bran concentration (50 g/L) with increased K2 HPO4 /KH2 PO4 concentration (2.275 g/L). Protease production obtained experimentally coincident with the predicted value and the model was proven to be adequate. Interestingly, the use of RSM increased the protease production by four times (7,000 U/mL) using a low-cost substrate and a culture time of 40 hr, as compared to the standard culture conditions. In the second part of this study, a H2 O2 -tolerant alkaline protease produced from B. invictae AH1 with a molecular mass of about 41 kDa, noted P3, was purified by successive steps of ultrafiltration, gel filtration and ion exchange chromatography. The K m and Vmax values of the purified protease using casein, as substrate, were about 4 mg/mL and 27 µM/min, respectively. The highest enzyme activity was found at pH 9.0 and a temperature of 60°C. In addition, the enzyme showed a quasi-total stability against H2 O2 (5% for 1 hr) and against most of the tested solid and liquid detergents, suggesting its eventual use in bio-detergent formulations.