The Importance of Developing a High Yield
of Product
European Antibody Congress Lyon, 3rd November 2005
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 20081
18 licensed products (16 since 1997), several of which are blockbusters
>150 in clinical trial, 15 identified in phase III2
PhRMA survey 20043 identifies MAbs as second largest
biopharma category in development after vaccines - 76 out of 324 ( 23% )
1. Reichert & Pavlou, Nature Reviews Drug Discovery,2004,3,383
2. Reichert et al. Nature Biotechnology 2005, 23,1073
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
5000 Liter Process for Protein Production
from Mammalian Cells
Kill System Utrafiltration 0.2 / 0.45µm IF Intermediate Filtration Intermediate Storage 2 - 8º c 50 Liter Fermenter 500 Liter Fermenter 5000 Liter Fermenter 0.2µm Filtration Concentration / Diafiltration Centrifuge 0.2µm Filtration Anion Exchange Concentration / Diafiltration 0.2µm Filtration 0.2µm Filtration Concentration or Dilutiuon Finished Goods 2 - 8º c Distributed to Customers Inoculum Grow-Up Final Filtration QC / QA Protein A Affinity Depth Filtration Media Prep Holding Tank
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 20061
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
20,000L Bioreactor & Add Tanks
Portsmouth, New Hampshire
Portsmouth, NH, USA
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) Large-Scale cGMP Production
Slough, England
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 ) R&D and Small-Scale Production
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
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
Enrichment for Highly Productive
Transfectants
Flow cytometric method
Rapid enrichment of transfectant pool prior to cloning
Enrichment of High Producing Cells
EP1415158 A, Lonza neutravidin bridge secreted antibody fluorochrome-labelled detection antibody biotinylated Protein A biotinylated-cell surfaceAnalysis 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
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
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. 1.0 – 3.6 g/l
Qp ( pg/cell/day ) 10 - 53
IVC (106 cells.h/ml) 900 – 3300
Max cell popn.density (106 cells/ml) 5 – 21
Qp is an important but not an exclusive determinant of
productivity – highest Qp does not always give highest volumetric titre
Prediction of bioreactor behaviour from
shake-flask model (GS-NS0)
0.0 0.4 0.8 1.2 1.6 2.0 antibody Qp Parameter V a lu e in re a c to r re la tiv e to shake-fl askCell Line Construction Method
Transfect host cells with vector
96 well plates, single colonies per well
200 – 300 cell lines
Quantitative productivity assessment
30 – 60 cell lines
Adapt to chemically defined medium 30 – 60 cell lines Static culture Preliminary quantitative assessment 5 - 10 cell lines
Fed-batch assessment of growth, productivity and product quality
Select cell lines to clone Clone
Suspension (Erlenmeyer flask) culture
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
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 1 1 0 1 0 0 0 1 0 0 2 0 0 3 0 0 4 0 0 T im e (h o u rs ) p H 7 .3 p H 7 .0
Batch vs. fed-batch operational modes
Batch: no additions
Feed solution
Fed-batch: small volume of
GS-CHO antibody titre improvements
since 1990*
old, titre = 0.04 g/L new, titre = 5.5g/L 0 2 4 6 8 10 12 0 48 96 144 192 240 288 336 384 432 480 528 576 T ime (h) V ia b le c e ll c o n c n (1 0 6 /m L ) 0 1 2 3 4 5 6 An tib o d y ( g /L )cells - old cells - new Ab - old Ab - new
Optimisation of a GS-CHO Process
Optimised a CHOK1 process using GS expressiontechnology 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
40 5520 New Clone 31 4301 Iteration 5 26 3560 Iteration 4 20 2829 Iteration 3 14 1917New cell line (CHOK1SV) 4 585 Iteration 2 2 334 Iteration 1 139 Original cell line
Fold Increase Antibody (mg/L)
GS-CHO optimisation; productivity
0 1000 2000 3000 4000 5000 6000 22H11 orig 22H11 v1 22H11 v2 LB01 v2 LB01 v3 LB01 v4 LB01 v5 CY01 v5 A n tibo dy ( m g /L ) 0.0 0.4 0.8 1.2 1.6 2.0 22H11 orig 22H11 v1 22H11 v2 LB01 v2 LB01 v3 LB01 v4 LB01 v5 CY01 v5Process development stage
S pec ifc pr od uc tion r a te ( p g /( ce ll. h) )
GS-CHO optimisation; growth
parameters
0 40 80 120 160 22H11 orig 22H11 v1 22H11 v2 LB01 v2 LB01 v3 LB01 v4 LB01 v5 CY01 v5 V iabl e c e ll conc ent ra tion ( 1 0 5/m L ) 0 1000 2000 3000 4000 22H11 orig 22H11 v1 22H11 v2 LB01 v2 LB01 v3 LB01 v4 LB01 v5 CY01 v5 IV C (1 0 6 c e ll.h /m L) 0 5 10 15 20 25 22H11 orig 22H11 v1 22H11 v2 LB01 v2 LB01 v3 LB01 v4 LB01 v5 CY01 v5Process development stage
P ro ce ss d u ra tio n ( d ay s)
Growth comparison:”Old” vs. “new” GS-CHO
cell lines growing in chemically defined medium
0 40 80 120 0 100 200 300 400 Time (h) V iab le cel l co n cen tr at io n ( 10 5 /m L ) 22H11 LB01
GS-CHO Product Accumulation
0 500 1000 1500 2000 2500 3000 3500 4000 0 500 1000 1500 2000 2500 3000Cumulative cell time (109 cell.h/L)
Prod ucgt co ncen tr at io n ( m g/ L)
22H11, iteration 1 22H11, iteration 2 LB01, iteration 2
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
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
Downstream Volumes for 2000l
Fermenter
Effect of Fermentation Titre on Buffer Demand
0 5,000 10,000 15,000 20,000 25,000 30,000 35,000 40,000 1 2 3 4 5 Fermentation Titre (g/L) Tot al Buf fe r Dem and (L)
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
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
