BioProduction 2005 Amsterdam Future Perspectives of Antibody Manufacturing John Birch Lonza Biologics
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 2008 1 18 licensed products (16 since 1997), several of which are blockbusters >150 in clinical trial, 15 identified in phase III 2 Of products in trial 42% are humanised and 28% fully human 1 PhRMA survey 2004 3 identifies MAbs as second largest biopharma category in development after vaccines - 76 out of 324 ( 23% ) Main therapeutic categories cancer and immunological 2 1. Reichert & Pavlou, Nature Reviews Drug Discovery,2004,3,383 2. Reichert et al. Nature Biotechnology 2005, 23,1073 3. PhRMA 2004 survey. Medicines in Development, Biotechnology
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
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
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 2006 1 from ca. 2 m.t. in 2004 Majority produced in batch / fed batch fermentation, some in perfusion Fermentation scales up to 20,000 litres 1. Gottschalk BioPharm International June 2005
20,000L Bioreactor & Add Tanks Portsmouth, New Hampshire
Portsmouth, NH, USA Large-Scale cgmp Production 60 miles from Boston s Airport 350,000 sq. ft. facility cgmp manufacture since 1996 1 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)
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 )
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 culture conditions, including physicochemical conditions and particularly feeding strategies Improved expression technology Stringent selection strategies to isolate high producers ( typically rare events ) High throughput screening Systems which are independent of position in genome Improved cell lines
GS-CHO antibody titre improvements since 1990 12 6 old, titre = 0.04 g/l Viable cell conc n (10 6 /m L ) 10 8 6 4 2 5 4 3 2 1 Antibody (g/l) new, titre = 5.5g/L 0 0 0 48 96 144 192 240 288 336 384 432 480 528 576 Time (h) cells - old cells - new Ab - old Ab - new ( Birch, in Protein Production by Biotechnology, Elsevier 1990 )
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
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
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) 139 334 585 1917 2829 3560 4301 5520 Fold Increase 2 4 14 20 26 31 40
Growth comparison: Old vs. new GS-CHO cell lines growing in chemically defined medium 120 Viable cell concentration (10 5 /ml) 80 40 22H11 LB01 0 0 100 200 300 400 Time (h)
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
Antibody purity by Coomassie Stained SDS PAGE analysis of In-Process Samples for a GS-CHO cell line Lane 1: Mol weight markers Lane 2: Inter assay control Lane 3: Cell culture supernatant Lane 4: Protein A eluate Main bands are antibody chains Minor bands are mostly host cell protein
Monoclonal Antibodies Upstream Progress Progress also being made in engineering cell lines to improve product characteristics e.g. glycoengineering to improve effector functions such as ADCC e.g. Glycart and Biowa Improved potency may reduce volumes required in the long term
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
Buffer Hold Upper Level
Purification 2.0M and 1.4M Columns
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 1 2 3 4 5 Fermentation Titre (g/l)
Where Next? Continued strong demand for monoclonal antibodies Mammalian cell culture processes likely to be important for forseeable future 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 Improvements in downstream technology
Where Next? Increased emphasis on engineering cells to alter product properties such as glycosylation Reduction in product heterogeneity? Manufacturing efficiency will increasingly be a factor influencing the initial design of the product
Examples of Carbohydrate Profiles for IgGs prepared in NS0 and CHO cell lines Terminal %digal %monogal %agal %sialylated Sugars Cell Line NS0 10.2 28.3 53.5 1.6 NS0 14.2 31.8 44.4 2.2 CHO 10.0 35.5 51.2 4.0 CHO 14.7 51.7 28.9 2.7 Glycosylation can vary from cell line to cell line with respect to detailed profile NS0 cells also produce a low level of 1,3 gal-gal and N glycolyl neuraminic acid
Where Next? Other technologies, especially microbial, are likely to become more significant especially for antibody fragments 15 MAbs in phase III identified by Reichert including: 3 single chain fragments 3 Fab fragments Whole antibodies expressed in bacteria ( in non glycosylated form ) and in yeast and fungi. Increasing potential to engineer desired glycosylation characteristics into microorganisms Transgenic plants, animals and eggs at comparatively early stage and face similar downstream challenges as other systems
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 Promise for other systems, especially microbial, in the longer term