BLT Stages

Abstract Bachelor Project  FBT 2019-2020: Validation of the calibration curves from enzyme activity obtained with NIR-spectrometry by means of an autoanalyzer

The determination of enzyme activity is a key analysis in the monitoring of the enzyme production process. Traditional techniques, such as manual or automated colorimetric analysis, require sample and reagent preparation and thus are time-consuming and demanding methods. In the need of a faster and cheaper analysis, near infrared spectroscopy was implemented for the enzyme activity measurement of fermentation samples.

The previously acquired near-infrared spectrometer is no longer supported by its manufacturer. Therefore, a new spectrometer, the FOSS NIRS DS2500, has been recently purchased as a replacement. As to apply this spectrometer in the daily laboratory routine, it needs to be validated.

Validation was carried out in two steps. In the first step, a minimum of hundred measurements were carried out of each type of enzyme and the resulting spectra were collected and sent to the manufacturing company of the spectrometer. The company then made a calibration curve for each type of analysed enzyme. In the second step, following the integration of the calibration curves in the software of the spectrometer, enzyme samples were measured with both the near-infrared method and an autoanalyzer colorimetric method. Finally, results between both methods were compared. Two other conditions were simultaneously studied: whether the FOSS NIRS DS2500 shows a noticeable improvement over the spectrometer that is due replacement (FOSS NIR 5000 DCA) and whether the use of different types of reflectors influences the analysis.

The study showed that the results were desirable with two out of the three analysed types of enzymes, as the average percentage difference between the enzyme activity measurements and the reference values is in both cases below the maximum established limit of 5 %. Overall, the FOSS NIRS DS2500 showed an improvement over the FOSS NIR 5000 DCA. The percentage difference is lower with the DS2500, with an average of 3,91 %. With the older spectrometer an average of 5,24 % is obtained. The use of different reflectors have shown to influence the measurements of enzyme activity, with reflector B showing slightly better results. The obtained average percentage difference with this reflector was of 6,10 %, and of 6,96 % with reflector A. Both of these are above the established maximum limit.

Although the FOSS NIRS DS2500 is not fully ready to be implemented in the laboratory, the prospects are very promising. In the near future, the already existing calibration curves of the analysed enzymes need to be updated with measurements only made with reflector type B. The calibration curves of the remaining enzyme types also need to be obtained and validated.

Abstract Bachelor Project 1 FBT 2018-2019: Rapid, automatic and innovative micro biology after validation of end products by means of Soleris vials in a BioLumix machine

Enzymes act as catalysts in many biochemical processes. In Genencor International - DuPont Bruges, three kinds of microorganisms are used to produce enzymes: bacteria, yeast and molds. It is extremely important that the final product, the enzymes, are completely uncontaminated  by these microorganisms. This can be tested in two ways: by carrying out a manual analysis on so-called ‘plates’ or by exercising an analysis by means of vials in a BioLumix machine. Until recently the latter analysis was performed by using BioLumix vials in Genencor International - DuPont plants. As these vials are no longer produced, Genencor International – DuPont started testing Soleris vials in order to decide whether these can be a trustworthy alternative in the future.

The aim of the present investigation, consequently, is to determine the absolute accuracy of the analyses of yeast, molds and TVC (total viable count) made by means of Soleris vials. This will be achieved by comparing these analyses with the results of the traditional manual analyses on plates. If these validation tests are positive, this will allow Genencor International - DuPont to make the necessary tests on the produced enzymes by means of the BioLumix machine again, with Soleris vials, which is performed a lot quicker and hence a lot cheaper than the manual analyses.The validation tests will be done on three samples of the same end product containing enzymes both in the BioLumix machine and on plates. The number of detected microorganisms by means of Soleris vials in the BioLumix machine should agree with the numerical values observed on the plates.

The results of the BioLumix tests indicate whether the dilution is larger or smaller than the dilution applied in the vial. The results of the manual analyses on the plate are expressed in numerical values, indicating the size of the recorded growth of microorganisms. If the values recorded by the BioLumix machine tally with those of the manual analyses, then the validation may be called successful. The results of this investigation and those in several other plants of Genencor International - DuPont have been collected and compared with each other in the United States. Already in the month of May of this year the conclusion was reached that the attempts to re-validate products on the BioLumix machine using the Soleris vials had been hardly successful and did not lead to great confidence in the Soleris vial type.

Consequently, Genencor International - DuPont has decided to suspend the validations for TAC, yeast and mold Soleris vials in the BioLumix machine immediately in all plants. The use of Soleris vials is considered to cause greater challenges than benefits presently. Genencor International - DuPont is now looking at The Global Microbiology Group for rapid microbiology methods as a solution of the problem.

Abstract Bachelor Project 2 FBT 2018-2019: Validation infrared technique using an auto analyzer

The analytical lab of Genencor International determines the enzyme activity of fermentation samples. The enzyme activity can be determined with a near infrared equipment and an autoanalyzer. A validation of a near infrared equipment was done during the internship period. This validation needs to happen every three years because the composition of the product can be changed. In addition, the validation ensures a more qualitative and stabile determination.

The validation checks if the calibration curve is still up to date for the samples which have been produced. At least three samples need to be measured for each fermentation with the near infrared device. The measured values are a prediction of the enzyme activity. The reference activity is determined from the same samples with an autoanalyzer. The validation gets approved as soon as the difference of both values is less than 5 %.

The near infrared device was validated for six different fermentations. The difference between the reference values and the predicted values is more than 5 % for five fermentations. This means that the five calibration curves should be updated. The other fermentation has been approved.

Abstract bachelor project FBT 2017-2018: Efficient microbiology using the BioLumix. Validation of enzyme-containing end products

Enzymes are biological catalysts that are significant in all essential biochemical processes. Because of their specific advantages, enzymes are increasingly used in various applications in the pursuit of a sustainable society. Due to the increasing demand, the production of enzymes is industrialized. In Genencor International-Dupont enzymes are produced from microorganisms.  The enzyme-containing end products must be free from contamination, impurities and the producing microorganism.  Therefore microbial testing is needed throughout the whole production process. The classic plate method is labor intensive, time consuming and the subjective interpretation of results can also be a disadvantage. The use of the BioLumix, a rapid microbiological method, can offer a solution because this method provides quicker and objective results.

Abstract bachelorproef 2016-2017: Introduction for the determination of the enzyme activity with the micro viscometer Lovis

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Abstract bachelorproef 2015-2016: Kalibratie en validatie IR techniek met behulp van een auto-analyser

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