APPLICATION NOTE 8*5 C Technologies, Inc. 9 A REPLIGEN COMPANY Author(s) Dawn M. Jones-Goldstein [email protected] 875 Chesterfield Parkway West Chesterfield, MO 63017 (636) 247-0127 Michael Jones [email protected] 875 Chesterfield Parkway West Chesterfield, MO 63017 (636) 247-2162 Sara Haydu [email protected] 685 Route 202/206 Bridgewater, NJ 08807 (908) 864-4972 Determination of Plasmid DNA Purity Ratio in Human Gene Therapy Products Using Slope Spectroscopy® Abstract Estimation of DNA purity in the presence of protein for gene therapy plasmid material is possible by exploiting the 260/280 UV absorbance ratio. This technique, however, has significant challenges when accurate quantification of DNA purity is required. The SoloVPE and its Slope Spectroscopy technique offer a new method to overcome these challenges, and to accurately measure DNA purity ratios in accordance with the Beer-Lambert law. DOC0178 Determination of Plasmid DNA Purity Ratio in Human Gene Therapy Products

£ 8 # C Technologies, Inc A REPLIGEN COMPANY Gene therapy is an exciting opportunity to change the way genetic diseases are treated. It has the potential to provide a lifelong solution to unmet medical needs. For these purposes, DNA purity is examined by testing the ratio of absorbance at 260 nm and 280 nm. This ratio is also known as the R value. Nearly all traditional spectrophotometers rely on fixed-pathlength UV-Vis absorbance readings. Several issues can arise from using this method, especially when dealing with the impurities of plasmids. Common impurities in plasmid DNA (pDNA) include...

APPLICATION NOTE Apparatus/Equipment SoloVPE Instrument, C Technologies, IN-VPE-SOLO5 ConfiRM Standard, 1.999 Abs/mm, CTechnologies catalog # MRM-PFI08-01-P10 ConfiRM Standard, 4.975 Abs/mm, CTechnologies catalog # MRM-PFI08-02-P10 ConfiRM Standard high, 23 Abs/mm, CTechnologies catalog # MRM-01-P1 Insulin, Sigma catalog # I0516-5ML Plasmid pCl-neo, project code 2019-1888, lot # MB2017459, Lake Pharma Plastic Sample Vessels OC0009-1-P50 Method & Results The R value is generally accepted as “pure” if the ratio is between 1.8 and 2.0. The ConfiRM standard was used to effectively demonstrate this...

APPLICATION NOTE £ 8 # C Technologies, Inc. • A REPLIGEN COMPANY Stock solutions of 1.0 mg/mL pCI-neo DNA plasmid and 1.0 mg/mL bovine serum insulin protein were prepared by diluting with water. A spectral scan of the stock solutions were taken from 200 nm to 360 nm. The absorbance profiles are demonstrated in Figure 1. System suitability testing was done with the SoloVPE to verify stock concentration at 1.0 mg/mL. Extinction coefficients used were as follows: Plasmid DNA s260 nm= 20 (^£) Bovine serum insulin 8280 nm = 0.965 (^0 cm-1 After confirming that the stock solutions were at 1.0 mg/mL,...

APPLICATION NOTE £ 8 # C Technologies, Inc. • A REPLIGEN COMPANY Table 1 (continued). Each purity level was analyzed by the SoloVPE. Triplicate slope measurements were obtained at 260 nm and 280 nm and averaged for a single reportable slope value. The R value was calculated for each level by slope ratio. Analysis The purity ratios obtained are plotted against their percent purity. The purity ratios obtained at each level correlate well with the theoretical ratio curve. The SoloVPE software provided the slope values at 260 nm and 280 nm simultaneously and then automatically calculated the ratio...

APPLICATION NOTE £ 8 # C Technologies, Inc. • A REPLIGEN COMPANY Table 2. Purity ratio accuracy Calculating the purity ratio, a theoretical ratio was determined for each level. As the protein and DNA absorbance is non-interfering, the absorption of light by the combination of both chromophores is additive and may be calculated by using the following equation: Slope RatiOi = ^ Slope Ratio^yj • 9 represents the volume fraction, • j represents the components (i.e., DNA or protein), and • i represents the wavelengths (i.e., 260 or 280 nm). ••• ••• DOC0178 Determination of Plasmid DNA Purity Ratio...

APPLICATION NOTE £ 8 # C Technologies, Inc. • A REPLIGEN COMPANY The theoretical and observed R values and percent error are compiled into Table 2. Of the 25 purity ratios obtained, the maximum observed error from theoretical was 1.56%. The average observed error was 0.25%. These results demonstrate a great accuracy and reproducibility at variety of purity ratios with a less than 2% deviation for all 25. Conclusion The current perception is that purity by UV 260/280 ratio is not useful, due to the availability of orthogonal assays with greater sensitivity. If values that exceed the typical variation...

APPLICATION NOTE References 1. O. Warburg and W. Christian (1942). Isolation and crystallization of enolase. Biochemische Zeitschrift, vol. 310, pp. 384–421. 2. D. M. Prazeres. (2011). Plasmid Biopharmaceuticals: Basics, Applications, and Manufacturing. John Wiley & Sons, Inc. 3. Pace, et. al. (1995). How to measure and predict the molar absorption coefficient of a protein. Protein Science. 4:2411-2423. 4. Ward, F. (1923). The Absorption Spectra of Some Amino Acids: The Possible Ring Structure of Cystine. Biochem J. 17(6):898-902 6. Leninger, A. L. (1975). Biochemistry, 2nd edition. Worth Publishers,...

APPLICATION NOTE £ 8 # C Technologies, Inc. W A REPLIGEN COMPANY 15. Standard Procedure for using ConfiRM® with the SoloVPE. (06 June 2018) DOC0138 Revision 00. C Technologies 16. Sawyer, Donald et. al. (1984) Chemistry experiments for instrumental methods. Wiley, New York. DOC0178 Determination of Plasmid DNA Purity Ratio in Human Gene Therapy Products