Selection of Cell Lines for Manufacturing Therapeutic Antibodies by Flow Cytometric Cell Sorting Andrew Racher Lonza Biologics
Structure of Talk 1. The Problem 2. Possible Solutions 3. Solution Chosen 4. Results 5/26/2005 / 2
The Problem Organisations developing therapeutic antibody drugs need to complete these activities as rapidly as possible whilst still meeting, for example: Economic criteria, e.g. exceeding a minimum productivity Regulatory criteria, e.g. clonal cell line How can the duration of cell line construction and development, a critical path activity, be reduced yet still meet these other criteria? 5/26/2005 / 3
Selection of high producing clonal GS-CHO cell lines Conventional cloning Transfection Suspension evaluation Cloning Suspension evaluation PSS 0 5 10 15 20 25 30 35 40 Time (weeks) To isolate high producing GS-CHO cell lines that fit manufacturing process need to evaluate transfectants in suspension culture before cloning Ensures good starting point for cloning Elapsed time is 40 weeks 5/26/2005 / 4
Possible solutions How can programme length be shortened? Duration can be reduced by moving straight from the transfection stage to the cloning stage Conventional cloning Transfection Suspension evaluation Cloning Suspension evaluation PSS 0 5 10 15 20 25 30 35 40 Time (weeks) 5/26/2005 / 5
Consequence of omitting a screening round (1) To ensure several cell lines with the desired growth and productivity characteristics are selected: screen more cell lines in each round Fewer rounds but, as p(good cell line) does not change, need to screen more cell lines per round No. of cell lines = p(good cell line) * no. screened 5/26/2005 / 6
Consequence of omitting a screening round (2) Alternatively, the pool of cell lines can be enriched for those with desired characteristics prior to each stage For example: Glutamine Synthetase (GS) Expression System indirectly enriches for high producers at transfection stage by use of the GS inhibitor MSX Use second method to enrich directly for high producers between transfection and cloning stages Antibody (mg/l) 350 300 250 200 150 100 50 0 25 µm 50 µm Selection conditions - MSX concentration 5/26/2005 / 7
Rapid enrichment of high producer sub-populations Number of flow cytometric-based assays developed that can identify and isolate high producing recombinant cell lines including: Gel Microdrop (Powell & Weaver, 1990, Biotechnol 8:333) Cellular affinity matrix (Holmes & Al-Rubeai, 1999, J Immunol Meth 230:141; Borth et al, 2001, Biotechnol Bioeng 71:266) Membrane trapping of secreted proteins (Brezinsky et al, 2003, J Immunol Meth 277:141) 5/26/2005 / 8
Approach taken with GS-cell lines 5/26/2005 / 9
Approach taken with GS-cell lines Affinity-matrix surface capture method for identifying high producing cells in transfectant pools Developed from method of Holmes & Al- Rubeai (1999) Fluorescence signal proportional to amount of secreted antibody Sort using FACS to enrich for high producers within population 5/26/2005 / 10
Affinity-matrix surface capture (AMSC): construction, capture and detection capture molecule (biotinylated Protein A) free, fluorochrome-labelled detection antibody neutravidin bridge captured, secreted antibody biotinylated-cell surface cell intracellular antibody EP1415158 A, Lonza 5/26/2005 / 11
Affinity-matrix surface capture (AMSC) biotinylated Protein A fluorochrome-labelled detection antibody neutravidin bridge secreted antibody biotinylated-cell surface cell EP1415158 A, Lonza 5/26/2005 / 12
Analysis of AMSC-labelled GS-CHO cells producing a recombinant antibody Fluorescent signals for antibody-producing GS-CHO cells were substantially higher than for non-producing cells 5/26/2005 / 13
The questions: Does high fluorescence correlate with high productivity? More accurately, specific production rate not product concentration Do cell lines generated by both the AMSC and conventional methods produce similar antibody concentrations in a model of the production process? 5/26/2005 / 14
Outline of process flows for AMSC and conventional methods 5/26/2005 / 15
Conventional cell line construction method Transfect host cells with vector 96 well plates, single colonies per well 200 300 cell lines Quantitative productivity assessment Static culture Suspension (Erlenmeyer flask) culture 30 60 cell lines Adapt to chemically defined medium 30 60 cell lines 5-10 cell lines Preliminary quantitative assessment Select cell lines to clone Fed-batch assessment of growth, productivity and product quality Clone 5/26/2005 / 16
AMSC-cloning cell line construction method Transfect host cells with vector Transfectant pools, T-flasks Analyse pools by AMSC method Bulk sorts Enriched transfectant pools Quantitative productivity assessment Clone Static culture Suspension (Erlenmeyer flask) culture Select cell lines 5-10 cell lines 30 60 cell lines Preliminary quantitative assessment 30 100 cell lines Quantitative 30 60 cell lines productivity assessment Adapt to chemically defined medium Fed-batch assessment of growth, productivity and product quality 5/26/2005 / 17
Comparison of AMSC-cloning and conventional methods - experimental approach (1) Conventional AMSC-cloning Starting point: wells with one colony Starting point: transfectant pools 40 wks duration 22 transfection + 18 cloning 31 wks duration Endpoint: clonal, high producing cell lines Endpoint: clonal, high producing cell lines 5/26/2005 / 18
Results 5/26/2005 / 19
Comparison of AMSC-cloning and conventional methods - experimental approach (2) Cell line CHOK1SV transfected with GS expression vector encoding HC and LC genes for a human IgG Conventional High producing non-clonal cell lines isolated from single, independent transfectant colonies Cell lines cloned to generate high producing clonal cell lines AMSC-cloning Transfectant pools enriched for high producing cell lines Pools for cloning chosen on basis of their SPR value SPR = antibody produced per cell over 48 hour period Productivities of cell lines compared at 4 points 5/26/2005 / 20
Does high fluorescence correlate with high productivity? Cell populations sorted from high and low signal regions Sorted populations expanded and SPRs determined The mean SPRs from the two regions are different (p = 0.05) High fluorescence correlates with high productivity Sorting enriched the population for high producers 5/26/2005 / 21
Conventional vs. AMSC-cloning: static culture (1) STATIC CULTURE SUSPENSION (Erlenmeyer flask culture) Conventional Transfect host cells with vector 200-300 cell lines 30-60 cell lines 30-60 cell lines 5-10 cell lines Single colonies per well Productivity assessment (quantitative) Adapt to chemicallydefined medium Preliminary productivity assessment (quantitative) Fed-batch assessment of growth, productivity and product quality Select cell lines for cloning STATIC CULTURE SUSPENSION (Erlenmeyer flask culture) AMSC-cloning Transfect host cells with vector Analysis by AMSC Enrich transfectant pools Clone 30 100 cell lines 30 60 cell lines 30 60 cell lines 5-10 cell lines Select cell lines for further analysis Pools in T-flasks Bulk sorts Productivity assessment (quantitative) Productivity assessment (quantitative) Adapt to chemicallydefined medium Preliminary productivity assessment (quantitative) Fed-batch assessment of growth, productivity and product quality 22 + cloning (18) = 40 wks 31 wks 5/26/2005 / 22
Conventional vs. AMSC-cloning: static culture (2) AMSC-cloning: populations from high signal region cloned; productivity of clones determined in small-scale static cultures Conventional: transfectants screened to remove low producers; productivity of survivors assessed in small-scale static cultures Are the mean antibody concentration values of the two sets of cell lines generated by the two methods equal? 5/26/2005 / 23
Conventional vs. AMSC-cloning: static culture (3) Distributions of productivities from the two methods are similar The mean values for antibody concentrations from the two methods are equal p = 0.17 5/26/2005 / 24
Conventional vs. AMSC-cloning: batch, suspension culture (1) STATIC CULTURE SUSPENSION (Erlenmeyer flask culture) Conventional Transfect host cells with vector 200-300 cell lines 30-60 cell lines 30-60 cell lines 5-10 cell lines Single colonies per well Productivity assessment (quantitative) Adapt to chemicallydefined medium Preliminary productivity assessment (quantitative) Fed-batch assessment of growth, productivity and product quality Select cell lines for cloning STATIC CULTURE SUSPENSION (Erlenmeyer flask culture) AMSC-cloning Transfect host cells with vector Analysis by AMSC Enrich transfectant pools Clone 30 100 cell lines 30 60 cell lines 30 60 cell lines 5-10 cell lines Select cell lines for further analysis Pools in T-flasks Bulk sorts Productivity assessment (quantitative) Productivity assessment (quantitative) Adapt to chemicallydefined medium Preliminary productivity assessment (quantitative) Fed-batch assessment of growth, productivity and product quality 22 + cloning (18) = 40 wks 31 wks 5/26/2005 / 25
Conventional vs. AMSC-cloning: batch, suspension culture (2) For both methods, antibody concentration in static culture used to rank cell lines. High ranked cell lines progressed Adapted to suspension culture growth Growth and productivity assessed in cultures operated in batch mode 5/26/2005 / 26
Conventional vs. AMSC-cloning: batch, suspension culture (3) Distributions of productivities from the two methods are not similar The mean values for antibody concentrations from the two methods are different Conventional 192 mg/l AMSC pool 136 mg/l 5/26/2005 / 27
Conventional vs. AMSC-cloning: fed-batch, suspension culture (1) STATIC CULTURE SUSPENSION (Erlenmeyer flask culture) Conventional Transfect host cells with vector 200-300 cell lines 30-60 cell lines 30-60 cell lines 5-10 cell lines Single colonies per well Productivity assessment (quantitative) Adapt to chemicallydefined medium Preliminary productivity assessment (quantitative) Fed-batch assessment of growth, productivity and product quality Select cell lines for cloning STATIC CULTURE SUSPENSION (Erlenmeyer flask culture) AMSC-cloning Transfect host cells with vector Analysis by AMSC Enrich transfectant pools Clone 30 100 cell lines 30 60 cell lines 30 60 cell lines 5-10 cell lines Select cell lines for further analysis Pools in T-flasks Bulk sorts Productivity assessment (quantitative) Productivity assessment (quantitative) Adapt to chemicallydefined medium Preliminary productivity assessment (quantitative) Fed-batch assessment of growth, productivity and product quality 22 + cloning (18) = 40 wks 31 wks 5/26/2005 / 28
Conventional vs. AMSC-cloning: fed-batch, suspension culture (2) Antibody concentration in batch culture used to rank cell lines High ranked cell lines progressed Growth and productivity assessed in cultures operated in fed-batch mode Key screening stage for selecting cell lines for use in cgmp manufacturing processes Erlenmeyer flask model of production bioreactor Uses same system of medium and feeds 5/26/2005 / 29
Conventional vs. AMSC-cloning: fed-batch, suspension culture (3) 5/26/2005 / 30
Conventional vs. AMSC-cloning: fed-batch, suspension culture (4) The mean antibody concentrations for sets of cell lines created by the two methods are equal p = 0.57 The mean Qp values for sets of cell lines created by the two methods are equal p = 0.65 Sets of cell lines generated by both the AMSC and conventional methods produced similar antibody concentrations in a model of the production process 5/26/2005 / 31
Conventional vs. AMSC-cloning: clonal cell lines (1) STATIC CULTURE SUSPENSION (Erlenmeyer flask culture) Conventional Transfect host cells with vector 200-300 cell lines 30-60 cell lines 30-60 cell lines 5-10 cell lines Single colonies per well Productivity assessment (quantitative) Adapt to chemicallydefined medium Preliminary productivity assessment (quantitative) Fed-batch assessment of growth, productivity and product quality Select cell lines for cloning STATIC CULTURE SUSPENSION (Erlenmeyer flask culture) AMSC-cloning Transfect host cells with vector Analysis by AMSC Enrich transfectant pools Clone 30 100 cell lines 30 60 cell lines 30 60 cell lines 5-10 cell lines Select cell lines for further analysis Pools in T-flasks Bulk sorts Productivity assessment (quantitative) Productivity assessment (quantitative) Adapt to chemicallydefined medium Preliminary productivity assessment (quantitative) Fed-batch assessment of growth, productivity and product quality 22 + cloning (18) = 40 wks 31 wks 5/26/2005 / 32
Conventional vs. AMSC-cloning: clonal cell lines (2) Antibody concentration used to rank cell lines in successive screening rounds Static then batch culture High ranked cell lines progressed Growth and productivity assessed in cultures operated in fed-batch mode Key screening stage Erlenmeyer flask model of production bioreactor 5/26/2005 / 33
Conventional vs. AMSC-cloning: clonal cell lines (3) 5/26/2005 / 34
Conventional vs. AMSC-cloning: fed-batch, suspension culture (4) Relationship between mean antibody concentration for daughter clones and parent does 95% CI for clone set include value of parent? Set A, no Set B, yes Only set A was different to its parent Relationship between mean specific production rate for daughter clones and parent does 95% CI for clone set include value of parent? Set A, yes Set B, yes Both sets were similar to their parents 5/26/2005 / 35
Conventional vs. AMSC-cloning: fed-batch, suspension culture (5) Relationship between the mean antibody concentrations for sets of cell lines created by the two methods Conventional = clone set B = AMSC clone set A p [ 0.05 Relationship between the mean specific production rates for sets of cell lines created by the two methods Conventional = clone set A = AMSC clone set B p [ 0.05 5/26/2005 / 36
Conventional vs. AMSC-cloning: fed-batch, suspension culture (6) When panels of cell lines were compared in the key screening stage (fed-batch culture), results indicate that: Cloning did not give systematic improvement in productivity characteristics between daughter clones and parent Cloning did not result in systematic improvement of productivity characteristics between clone sets A and B compared and the cell line sets Conventional and AMSC Overall, the data do not indicate a systematic improvement in productivity between clonal cell lines generated by the conventional method and the AMSC-cloning method Exceptions can be found 5/26/2005 / 37
Impact of using AMSC-cloning approach Both methods generated a panel of cell lines having similar productivities Enrichment of transfectant pool before cloning has shortened screening step Time saving = 9 weeks Conventional cloning Transfection Suspension evaluation Cloning Suspension evaluation PSS AMSC-cloning Transfection AMSC-sort & expansion Cloning Suspension evaluation PSS 0 5 10 15 20 25 30 35 40 Time from start (weeks) 5/26/2005 / 38
Summary Cell line construction and selection methods were run in parallel using a conventional protocol and a flow cytometric protocol Cytometric method enriched cell populations for high producers For the key selection stage, evaluation in fed-batch suspension culture, mean antibody concentration and mean specific production rate were similar The productivities of the panels of candidate cell lines were generally similar AMSC-cloning method reduces timeline by 9 weeks Potential for further improvement if qualify FACS for single cell sorting 5/26/2005 / 39
Acknowledgements Cell Culture Process Development Group, LB, Slough Assay Development Group, LB, Slough 5/26/2005 / 40