The Importance of Developing a High Yield of Product

Transcription

1 European Antibody Congress Lyon, 3 rd November 2005 The Importance of Developing a High Yield of Product John Birch, Lonza Biologics plc

2 Monoclonal Antibodies A Success Story Fastest growing segment of the pharmaceutical market Sales forecast to increase from $5.4b in 2002 to $16.7b in licensed products (16 since 1997), several of which are blockbusters >150 in clinical trial, 15 identified in phase III 2 PhRMA survey identifies MAbs as second largest biopharma category in development after vaccines - 76 out of 324 ( 23% ) 1. Reichert & Pavlou, Nature Reviews Drug Discovery,2004,3, Reichert et al. Nature Biotechnology 2005, 23, PhRMA 2004 survey. Medicines in Development, Biotechnology

3 Monoclonal Antibodies How Are They Made Licensed products all made in mammalian cell culture 10 produced in CHO 8 produced in lymphoid cells esp. NS0 and Sp2/0 Majority produced in batch / fed batch fermentation, some in perfusion Fermentation scales up to 20,000 litres Downstream based on chromatography Protein A used in majority of cases followed by two to three additional steps; ion exchange and sometimes HIC, size exclusion Virus removal / inactivation steps included

4 5000 Liter Process for Protein Production from Mammalian Cells Media Prep Depth Filtration Centrifuge Inoculum Grow-Up Kill System 50 Liter Fermenter 500 Liter Fermenter 5000 Liter Fermenter Utrafiltration 2-8º c Concentration / Diafiltration 0.2 µm Filtration 0.2 µm Filtration Protein A Affinity Intermediate Storage 0.2 / 0.45 µm IF Intermediate Filtration Holding Tank Concentration or Dilutiuon 2-8º c Anion Exchange 0.2 µm Filtration Concentration / Diafiltration 0.2 µm Filtration Final Filtration QC / QA Finished Goods Distributed to Customers

5 Monoclonal Antibodies The Quantities Frequently used at much higher doses than other biopharm proteins, leading to large volume demands 10s to 100s of kg per year and possibly tons in the future Predicted that demand will have increased to ca. 6 m.t. by from ca. 2 m.t. in 2004 Increased demand has been a driver for: Increased capacity worldwide Increase in scale of reactors (up to 20,000 litres) to realise economies of scale Development of more productive processes 1. Gottschalk BioPharm International June 2005

6 20,000L Bioreactor & Add Tanks Portsmouth, New Hampshire

7 Portsmouth, NH, USA Large-Scale cgmp Production 60 miles from Boston s Airport 350,000 sq. ft. facility cgmp manufacture since x 2,000 l airlift 2 x 5,000 l airlift 2 x 1,500 l perfusion 3 x 20,000 l stirred 1 x 20,000 l stirred (2006)

8 Slough, England R&D and Small-Scale Production 10 miles Heathrow airport R&D facility incl. pilot plant cgmp manufacture : Disposable bioreactors 20 l to 400 l 2 x 200 l airlift 2 x 2,000 l airlift 500 l stirred ( 2006 )

9 Monoclonal Antibodies Upstream Progress Titres of 1 to 4 g/l now typical and 10g/l probably achievable Titres of 5.5 g/l (Lonza ) and 6.1 g/l (Abbott) reported for CHO, 5.1g/l for NS0 Improvements have come from two areas of development Improved expression technology Highest Qps have not changed (tens of pg/cell/day) but can be achieved routinely and much more rapidly Stringent selection strategies to isolate high producers (typically rare events) High throughput screening Systems which are independent of position in genome Improved cell lines Improved culture conditions, including physicochemical conditions and particularly feeding strategies

10 Glutamine synthetase (GS) gene expression system Expression vector encoding product gene(s) plus GS gene, allowing synthesis of glutamine an essential nutrient Only cells with GS gene (and hence product gene) survive Increase selection stringency - use weak promoter on GS gene - selects for rare integration into transcriptionally efficient sites in genome GS is inhibited by methionine sulphoximine (MSX) which can be used to increase stringency of selection Linked product gene driven by strong promoter (hcmv) to give high expression High productivity without amplification

11 Enrichment for Highly Productive Transfectants Flow cytometric method Rapid enrichment of transfectant pool prior to cloning Avoids time consuming screening process in small scale culture

12 Enrichment of High Producing Cells biotinylated Protein A fluorochrome-labelled detection antibody neutravidin bridge secreted antibody biotinylated-cell surface EP A, Lonza

13 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 Collect cells with high fluorescence

14 Challenges in Cell Line Creation Screening methods tend to focus on Qp Individual transfectants show enormous phenotypic variability, not just in Qp but also in growth characteristics Challenge is to predict manufacturing behaviour of cell lines at very early stage and select clones with appropriate Qp and growth characteristics Use fed batch shake flask culture

15 Variation in growth characteristics seen in shake flask screen of GS-CHOs Overall ranges for four groups of transfectants (four antibodies) Ten fed shake flasks per group All cultures producing > 1g/l antibody Product concn g/l Qp ( pg/cell/day ) IVC (10 6 cells.h/ml) Max cell popn.density (10 6 cells/ml) 5 21 Qp is an important but not an exclusive determinant of productivity highest Qp does not always give highest volumetric titre

16 Prediction of bioreactor behaviour from shake-flask model (GS-NS0) 2.0 Value in reactor relative to shake-flask antibody Parameter Qp

17 Cell Line Construction Method Transfect host cells with vector 96 well plates, single colonies per well cell lines Quantitative productivity assessment Static culture Suspension (Erlenmeyer flask) culture cell lines Adapt to chemically defined medium cell lines 5-10 cell lines Preliminary quantitative assessment Select cell lines to clone Fed-batch assessment of growth, productivity and product quality Clone

18 Monoclonal Antibodies Upstream Progress Titres of 1 to 4 g/l now typical and 10g/l probably achievable Titres of 5.5 g/l (Lonza ) and 6.1 g/l (Abbott) reported for CHO, 5.1g/l for NS0 Improvements have come from two areas of development Improved expression technology Highest Qps have not changed (tens of pg/cell/day) but can be achieved routinely and much more rapidly Stringent selection strategies to isolate high producers (typically rare events) High throughput screening Systems which are independent of position in genome Improved cell lines Improved culture conditions, including physicochemical conditions and particularly feeding strategies

19 Effect of Culture ph Reduction of culture ph for a protein-free (chemically defined medium) GS-NS0 process Increased maximum viable cell concentration Increased culture duration Increased integral viable cell hours Increased productivity 590 mg/l compared with 240 mg/l Time (hours) ph 7.3 ph 7.0

20 Batch vs. fed-batch operational modes Feed solution Batch: no additions Fed-batch: small volume of concentrated nutrient solution added

21 GS-CHO antibody titre improvements since 1990* 12 6 old, titre = 0.04 g/l Viable cell conc n (10 6 /m L ) Antibody (g/l) new, titre = 5.5g/L Time (h) cells - old cells - new Ab - old Ab - new * Birch, in Protein Production by Biotechnology, Elsevier 1990

22 Optimisation of a GS-CHO Process Optimised a CHOK1 process using GS expression technology to produce a monoclonal antibody ( cb72.3 ) Improved culture conditions especially feeds and physicochemical conditions Antibody genes transfected into improved variant of CHOK1 ( CHOK1SV ) New host (CHOK1SV ) grows spontaneously in suspension in chemically defined, protein free, medium without hydrolysates

23 Process Optimisation for a GS-CHO cell Line Process Original cell line Iteration 1 Iteration 2 New cell line (CHOK1SV) Iteration 3 Iteration 4 Iteration 5 New Clone Antibody (mg/l) Fold Increase

24 GS-CHO optimisation; productivity Antibody (mg/l) H11 orig 22H11 v1 22H11 v2 LB01 v2 LB01 v3 LB01 v4 LB01 v5 CY01 v5 Specifc production rate (pg/(cell.h)) H11 orig 22H11 v1 22H11 v2 LB01 v2 LB01 v3 LB01 v4 LB01 v5 CY01 v5 Process development stage

25 GS-CHO optimisation; growth parameters 160 Viable cell concentration (10 5 /ml) H11 orig 22H11 v1 22H11 v2 LB01 v2 LB01 v3 LB01 v4 LB01 v5 CY01 v IVC(10 6 cell.h/ml) H11 orig 22H11 v1 22H11 v2 LB01 v2 LB01 v3 LB01 v4 LB01 v5 CY01 v5 25 Process duration (days) H11 orig 22H11 v1 22H11 v2 LB01 v2 LB01 v3 LB01 v4 LB01 v5 CY01 v5 Process development stage

26 Growth comparison: Old vs. new GS-CHO cell lines growing in chemically defined medium 120 Viable cell concentration (10 5 /ml) H11 LB Time (h)

27 GS-CHO Product Accumulation Producgt concentration (mg/l) Cumulative cell time (10 9 cell.h/l) 22H11, iteration 1 22H11, iteration 2 LB01, iteration 2 LB01, iteration 3 LB01, iteration 4

28 Protein-free chemically defined media Increasing emphasis from regulatory authorities on removal of animal-derived raw materials from antibody production processes Reduces risk of introducing adventitious agents and other contaminants Makes process optimization easier if ill defined additives such as serum and hydrolysates are avoided Cost benefits Reduces protein load in purification

29 Downstream Issues As upstream titres increase, downstream processing volumes increase in direct proportion to titre At current titres, buffer volumes can be an order of magnitude greater than upstream reactor Downstream becomes an increasing proportion of total costs; expensive chromatography steps including Protein A Growing interest in addressing downstream issues e.g More efficient chromatographic steps Fewer unit process steps Novel technologies e.g. membrane adsorbers

30 Buffer Hold Upper Level

31 Purification 2.0M and 1.4M Columns

32 Downstream Volumes for 2000l Fermenter Total Buffer Demand (L) 40,000 35,000 30,000 25,000 20,000 15,000 10,000 5,000 0 Effect of Fermentation Titre on Buffer Demand Fermentation Titre (g/l)

33 Where Next? Mammalian cell culture processes likely to be important for forseeable future ( microbial systems showing promise for long term ) Continued improvements to the fermentation process Increased emphasis on improving host cell lines for improved productivity finding the bottlenecks downstream of transcription Use of knowledge from omics studies to inform process design and cell engineering Continuing improvements to media and feeds Manufacturing efficiency will increasingly be a factor influencing the initial design of the product Increased product potency may reduce volumes required Improvements in downstream technology

34 Summary Large scale manufacturing technology for MAbs has developed rapidly in recent years (100x increase in titres in ca. 15 years) Improvements likely to continue based on further improvements to mammalian cell systems (cell lines and fermentation conditions) Downstream processing becoming an increasingly important area of focus for efficiency improvements