
Design of Experiment Principles to Optimize Benzonase® Endonuclease in Viral Gene Therapy Applications

Setting up the DoE
In order to establish an effective DoE, it is important to define constant factors, input variables with center point starting conditions and output parameters, first (Table 1). The constant factors are key parameters that will remain constant throughout the DoE experiments. For example, the concentration of magnesium, which should be held at 2mM as it is necessary for proper function of the enzyme. Importantly, prior to initiation of the DoE, the average DNA load of the input sample material should be determined in order to calculate the required amount of enzyme. Also note that the enzyme activity in U/μL is provided on the Certificate of Analysis for each lot and can be used to calculate the volume of Benzonase® endonuclease using the equation in Table 1, center point starting conditions. The center point conditions provide users with a point of reference from which parameters can be altered to determine the relationship between the input and output factors. Basing DoE experiments on enzyme activity, instead of protein concentration, ensures reproducible DNA digestion.Parameters | |
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Constant Factors | Average DNA load Equipment: incubation vessel and mixing process Upstream material: pH and salt concentration [Mg2+] = 2mM |
Input Variables | Benzonase Concentration Incubation Time Incubation Temperature |
Center Point Starting Conditions | Benzonase Concentration:![]() Incubation Time: 2 hours Incubation Temperature: 21°C |
Output Variables | DNA content of >100 bp fragments Product Integrity |
