Transient production of recombinant proteins using the MEXi system

Transcription

1 Transient production of recombinant proteins using the MEXi system Dennis Niermeier IBA GmbH Dennis Niermeier 216

2 Small amounts (mg range) of protein are required in a short time for many experiments and applications. crystallization antibody screening protein-protein interaction studies in vivo studies assay development high throughput applications 2

3 Transient expression generates protein in mg range in short time. Optimization of the production process can require much effort. cloning vector screening for selected cell line transfection expression purification analysis selection of transfection medium and reagent development and optimization of a transfection protocol selection of a (suspension) cell line medium selection (serum free), adaptation of the cell line optimization of expression selection of purification matrix/strategy optimization of purification (protein dependent) specific detection of the protein of interest (POI) e. g. in supernatant, western blot, ELISA, POI specific antibodies 3

4 IBA s system MEXi (mammalian expression system from IBA) enables a platform suited for the production from gene to protein, independent of protein properties. cloning cloning - vectors - free choice of tag and terminus - TOP 1 E. coli transfection MEXi system transfection - suspension HEK-293 EBNA cells - economic transfection medium (CD) - linear PEI - optimized protocols expression cultivation/ expression - economic cultivation medium (CD) - optimized process - reduced biotin concentration purification purification - protein independent Strep-tag purification - one-step purification with high purity analysis analysis - Strep-tag specific detection (western blot, ELISA) 4

5 The pdsg vector was designed to make use of MEXi cell s properties. small pdsg vector for high plasmid yield including orip enhanced nuclear import and transcription due to orip and EBNA gene ColE1 ori and ampicillin resistance for propagation in E. coli vector only contains IP free elements no license fees 5

6 cell density [x 1 5 cells/ml] viability [%] MEXi-293E cells are robust for changes in cultivation over a long time, maintaining an average viability of 98 %, an average generation time of 25 h and reach a maximum cell concentration 15 x 1 6 cells/ml day period without subcultivation removal for transfection days cell density viability

7 DNA [mg/l] The transfection efficiency is mainly influenced by the PEI concentration. Transfection efficiencies up to 76 % (GFP positiv cells) were measured. Results of central composite design (CCD): GFP positive cells [%] economical approach mid density/economical approach high GFP approach 1 x 1 6 cells/ml 1.4 x 1 6 cells/ml 1.8 x 1 6 cells/ml PEI [mg/l] selected for further experiments 7

8 yield [mg/l] A DNA concentration of 1.5 mg/l, a PEI concentration of 4.5 mg/l and a cell density of 1.5 x 1 6 cells/ml led to the highest protein yield (145 mg/l) in the experiment DNA [mg/l] PEI [mg/l] Cell density [x 1 6 cells/ml] SEAP yield 8

9 yield [mg/l] Only the dilution after transfection seems to have a significant impact on the expression. The protocol was optimized to develop the most robust process. half-normal plot for yield, alpha=.1 main effects plot for yield settings for the final protocol 9

10 SEAP yield [mg/l] GFP postive cells [%] mean fluorescence intensity [%] MEXi-293E cells have a constant expression over several passages Passage 34 Passage 67 Passage 34 Passage 67 Passage 34 Passage 67 1

11 GFP positiv cells [%] mean fluorescence intensity MEXi-293E cells achieve high expression levels due to orip in pdsg. GFP mean fluorescence of cells transfected with pdsg is 3.4 fold higher compared to pcdna3 transfected cells 48 h post transfection pdsg pcdna3 pdsg pcdna3 11

12 MEXi was successfully tested for different recombinant proteins (up to 318 mg/l) and provides high protein purity. SEAP (143 mg/l) mab (96 mg/l) customer protein (318 mg/l) marker, supernatant (line 1) and elution fraction (line 2) of a SEAP and monoclonal antibody expression using MEXi system marker, elution of a customer protein expressed with the MEXi system 12

13 MEXi keeps up with leading expression systems. competitive expression system (CES) SEAP yield [mg/l] MEXi seeding cell density [x 1 6 cells/ml] DNA [mg/l] 1.75 medium Freestyle F17 MEXi media medium list price [EUR] labor compared to CES costs/mg SEAP (compared to CES) 1.72 costs for 1 L expression (compared to CES) 1.75 license fee on request none 13

14 The use of the MEXi system and the IBA platform reduces time and costs. suspension cells, chemical defined medium and powerful vector are balanced with the other IBA products (e.g. StarGate, purification matrices) or can also be used independently affordable media affordable medium allows cost effective PEI transfection low biotin concentration in the media makes Strep-tag purification more cost efficient elution under native conditions no need to optimize purification because it is based on the Strep-tag and not on varying protein properties purity usually is > 95 % due to effective Strep-tag purification license free expression system (for research and commercial use) use of the same material for all protein productions reduces the amount of different materials methods and protocols 14

15 If you like, visit my Poster # 4: MEXi expression system Development of an affordable mammalian transient expression system More information about IBA: twitter.com/ibalifesciences linkedin.com Facebook 15