Vpeljava tehnologije kapljične digitalne verižne reakcije s polimerazo v razvoj bioloških zdravil

Dermota, Tjaša (author), Doljak, Bojan (mentor), Vogelsang, Matjaž (co-mentor)

Kapljična digitalna verižna reakcija s polimerazo (ddPCR) je novejša oblika digitalne verižne reakcije s polimerazo (dPCR). Metoda temelji na tvorbi približno 20.000 kapljic, ki nastanejo v emulziji voda v olju (V/O), verižni pomnožitvi s polimerazo in končni absolutni kvantifikaciji kapljic s prisotnim pomnoženim produktom. Metoda je uporabna na številnih področjih, v okviru magistrske naloge pa smo želeli preveriti, če jo lahko uporabljamo tudi za razvoj rekombinantnih bioloških učinkovin v biofarmacevtski industriji. Pri izbiri ustreznih klonov igra ključno vlogo karakterizacija števila kopij, integritete in stabilnosti transgena. V podjetju Lek d.d. v ta namen uporabljajo verižno reakcijo s polimerazo v realnem času (qPCR) in metodo prenosa po Southernu, ki pa ju želijo nadomestiti z novejšo metodo – ddPCR. Metodo ddPCR smo najprej optimizirali in nato preverili njeno uporabnost za genetsko karakterizacijo klonov ovarijskih celic kitajskega hrčka (CHO), uporabljenih v proizvodnji rekombinantnih proteinov, zlasti monoklonskih protiteles. Preverili smo analitske parametre ddPCR pri določanju števila kopij in integritete vstavljenih transgenov, izražanja lahke in težke verige protiteles v celičnih linijah in možnost uporabe ddPCR za kvantifikacijo rezidualne DNA v različnih stopnjah čiščenja zdravilne učinkovine. Določili smo široko dinamično območje, ki ga pokriva ddPCR tako za določanje koncentracije DNA (4 – 8000 kopij/μL), kot tudi mRNA (8 – 20.000 kopij/μL). Gre za visoko občutljivo metodo, saj je limita detekcije 1,5 – 3 kopije tarčnega gena/μL pri vzorcih DNA, ter 1,2 kopij/μL pri RNA vzorcih. Variabilnost metode pri kvantifikaciji DNA se giblje med 10 in 15 % in je nekoliko višja pri uporabi barvila EvaGreen v primerjavi s hidrolizirajočimi sondami. DdPCR smo primerjali z uveljavljenimi metodami in ugotovili, da je metoda primerna za določanje števila kopij in izražanja transgenov ter rezidualne DNA. Pri preverjanju integritete vstavljenih transgenov je ddPCR izkazala 80 % občutljivost, 88 % specifičnost in 82 % točnost glede na metodo prenosa po Southernu. Pri določanju stabilnosti vstavljenih transgenov je ddPCR izkazala 40 % občutljivost, 100% specifičnost ter 70 % točnost glede na prenos po Southernu. V razvoju bioloških zdravil je metoda ddPCR primerna za preverjanje integritete transgenov in določanje števila kopij pri presejanju klonov, saj je hitrejša in v primeru določanja števila kopij tudi točnejša. Za uporabo pri določanju stabilnosti pa bi ddPCR potrebovala še nadaljnjo optimizacijo.

• Language: Slovenian
• Keywords: ddPCR, celice CHO, integriteta transgena, stabilnost klona, rezidualna DNA, ekspresija transgena, optimizacija ddPCR
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-124802
• Publication date in RUL: 19.02.2021

Secondary language

• Language: English
• Title: Implementation of droplet digital polymerase chain reaction technology in biological drugs development

Abstract

Droplet digital polymerase chain reaction (ddPCR) is a new form of digital polymerase chain reaction (dPCR). The method is based on the formation of about 20,000 droplets formed in a water-oil emulsion, a polymerase chain reaction and final absolute quantification of droplets with a multiplied product. Although the method is applicable in many fields, the scope of the master's thesis was to check if it can also be used in the development of biological substances in the biopharmaceutical industry. For the selection of suitable clones, characterization of copy number, integrity and stability of the transgene plays an important role. For this purpose, the pharmaceutical company Lek d.d. uses the Southern blot and real-time polymerase chain reaction (qPCR), which is sought to be replaced with the newer ddPCR method. The ddPCR method was first optimised and then tested for its suitability for the genetic characterization of Chinese Hamster Ovary (CHO) clones, used for the production of recombinant proteins - mostly monoclonal antibodies. We tested the analytical parameters of ddPCR for determination of copy number and integrity of inserted transgenes, expression of the light and heavy chain of the antibody in cell lines and the possibility of using ddPCR to quantify residual DNA at different stages of drug purification. DdPCR covers a wide dynamic range of concentration determination for both DNA (4 – 8000 copies/μL) and mRNA samples (8 – 20,000 copies/μL). This is a highly sensitive method, since the detection limit is 1.5 – 3 copies of the target gene/μL in the case of DNA samples and 1.2 copies/μL in the case of RNA samples. The variability of the method for DNA quantification is between 10 and 15% and is slightly higher for the EvaGreen dye compared to TaqMan probes. We compared ddPCR with established methods and demonstrated that ddPCR is a suitable method for determination of transgene copy number, transgene expression and residual DNA concentration. For determination of integrity of inserted transgenes, ddPCR displays a 80% sensitivity, 88% specificity and 82% accuracy compared to the Southern blot method. For the determination of stability of inserted transgenes ddPCR showed a 40% sensitivity, 100% specificity, and 70% accuracy, compared to Southern blot. Thus ddPCR represents a suitable method for determination of transgene integrity and copy number during the clone screening process, as it is faster and in the case of copy number determination also more accurate. To be used for transgene stability determination, ddPCR needs further optimisation.

• Keywords: ddPCR, CHO cells, transgene integrity, clone stability, residual DNA, transgene expression, ddPCR optimisation