strategies for the purification of high titre, high volume
TRANSCRIPT
Strategies for the purification of high titre, high volume mammalian cell culture batches
Martin P. Smith.
LONZA Biologics plc, 228 Bath Road, Slough, SL1 4DX.
Presented at,Recovery & Purification.BioProcess International European Conference and Exhibition.Berlin, April 2005.
15/04/2005 / 2
Overview
Review of Fermentation process productivity increasesCurrent status
Challenges presented by high titre, high volume batches
Strategies available for dealing with high-titre, high-volume batches
Opportunities within current downstream processing technologiesChromatographyBuffer supplyUltrafiltration
Importance of timely process developmentSimple tools & methods for process development and scale-up
15/04/2005 / 3
LONZA Biologics manufacturing capacity
LONZA currently operates monoclonal antibody facilities in both the US and UK
UK Manufacturing-focused on rapid supply of clinical phase materialVarious disposable units (Wave)2x2000L ALF2x200L ALFAdditional capacity planning exercise underway
US Manufacturing-focused on large-scale late phase & in-market supply
2x5000L ALF1x2000L ALF2x1500L perfusion (dedicated)3x20,000L STR growing to 4 in 2006
15/04/2005 / 4
Biopharmaceutical Production Challenges
Increased demand for cGMP manufacturing capacity at all scales.
Increase in product approvals and marketing extensions driving ademand for large scale capacity (>5000L reactor volume)
Strong demand for small scale capacity driven by full customer pipelines and need for early phase toxicology & clinical study material.
Growing political and ethical pressures to control and reduce drug development time-to-market and production costs.
Cell Line Construction cGMP
Cell Banking
Process Development
Scale-Up &Pilot Prod.
cGMPManufacture
15/04/2005 / 5
Advances in Mammalian Cell Culture Process Productivity
0 5 10 15 20 25
1000
2000
3000
4000
5000
6000
Iteration 1 (22H11) Iteration 2 (22H11) Iteration 2 (LB01) Iteration 3 (LB01) Iteration 4 (LB01) Iteration 5 (LB01) Iteration 5 (CY01)
Pro
duct
Acc
umul
atio
n (m
g/L)
Fermentation Time (days)
Fermentation productivity is rising RAPIDLY.
Advances in cell culture realising higher titres
Already experiencing 4-5g/L batches after 14days in cGMP reactors
15/04/2005 / 6
Current Capacity Crunch
Much of today’s production capacity for in-market supply designed for low to modest productivities of monoclonal antibodies
Resulted in requirement for multiple large volume reactors (4-6 x 12-20kL) for single purification trains.
In-house BioPharma capacity can be optimised for specific medium to low risk products
Contract Manufacturing capacity must remain reflective of wider industry status to capture current and near term business
Rate of technology development in mammalian expression outpacingdesign-and-build timelines for new facilities
Industry requires low risk, low capital and immediate solutions to purifying high productivity batches across all scales.
15/04/2005 / 7
Identifying bottlenecks in downstream processing
Situation for CMO’s is more complicated than single product facilities
Business risk managed through large customer base
Each product differs widely despite generic/platform technologies
Fermentation productivitiescolumn capacities, number of column stepsDiffering degrees of process intensification
Increasing titres poses significant challenges for:
Throughput (speed of purification)Economics (Batch cost, cost of goods)
15/04/2005 / 8
Strategies to alleviate downstream processing bottlenecks
1. Invest in development of novel technologies
Membrane Adsorbers (PnA capture and contaminant removal)Mimetic ligandsMonolithic chromatography supportsUV for virus inactivation
2. Invest in development of conventional technologies
Centrifugation2 phase separationsSolvent extractionPrecipitationCrystallisation
15/04/2005 / 9
Factors complicating selection of strategy
5g/L titres are here now!
“Novel” technology and re-development of “conventional” technologies may not help in the near term
Both involve significant investment of capital to refit existing facilities
Need to “sell” technology improvementsInternally across companyExternally to clients
Technology solutions must be scalable and generic
15/04/2005 / 10
3. Maximise utilisation of existing assets.Process OptimisationDownstream Yield Improvement ProgramsProcess IntensificationReduce processing timeEliminate non-processing timeReduce start-up and turn around/change over times
4. Drive process excellence across businessLEAN/Six Sigma/Quality systems
Further Strategies to alleviate downstream processing bottlenecks
15/04/2005 / 11
0.1-1g
Constructexpression
vector
ConstructCell Line
DevelopManufacturing
Process
Non GMPPilot Run
GMPProduction
Cell culturesupernatants
Protein Apurifiedproduct
Researchgrade fully
purifiedproduct
Material fortoxicologystudies,
referencestandard,
stability studies
Clinical TrialSupply
MaterialSupply
TypicalQuantities 10-50mls 10-50mgs 10-100g
0.1kg tomulti-kgs
Typical Development Program
Speed to Clinic for Early Phase material ensured through use of generic processesFast Track Downstream development characterised by aggressive timelines
15/04/2005 / 12
Focus of presentation
Bottlenecks and Production Economics change markedly as a function of scale.
Key bottlenecks and areas for cost/throughput optimisation:
Protein A Capture
Buffer Supply
Ultrafiltration Optimisation
15/04/2005 / 13
Example of 2000L reactor titre increase.
For same purification process:
Chromatography elution tank volume constraints
Ultrafiltration tank volume capacity
Virus filtration throughput constraints
Increased demand for buffers
Effect of fermentation titre on intermediate process volumes
Process Volume vs Concentration
0
500
1000
1500
2000
2500
Har
vest
CC
S
PnA
Elu
ate
VI E
luat
e
UF
1Pr
oduc
t
Q F
T
UF
2Pr
oduc
t
VR
FPr
oduc
t
SP P
rodu
ct
UF
3Pr
oduc
t
Pro
cess
Vol
(L)
0
5
10
15
20
25
30
Proc
ess
conc
(g/L
)
Lg/L
Process Volume vs Concentration
0
500
1000
1500
2000
2500
Har
vest
CCS
PnA
Elu
ate
VI E
luat
e
UF
1P
rodu
ct
Q F
T
UF
2P
rodu
ct
VR
FP
rodu
ct
SP
Prod
uct
UF
3P
rodu
ct
Pro
cess
Vol
(L)
0
5
10
15
20
25
30
Proc
ess
conc
(g/L
)
Lg/L
1g/L
5g/L
15/04/2005 / 14
Process Development Case Study 1
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 5Fermentation Titre (g/L)
Tota
l Buf
fer D
eman
d (L
)
Reducing Buffer loading during downstream processing
10-15 % of buffer Demand is derived from Protein A step
2000L reactor titre increase.
15/04/2005 / 16
Buffer Concentrates and In-Line Dilution
Possible to minimise Buffer Make-up and Hold requirements through in-line dilution
Example: Protein A Equilibration, Post Load Wash Buffer– 50mM Glycine Glycinate, 250mM Sodium Chloride, pH 8.0
C
H
HH2N
COO-Na+
C
H
HH3N+
COO-
C
H
HCl-H3N+
COOH
Glycine Hydrochloride
(AA+1)
Isoelectric Glycine
(free base)
(AA0)
Sodium Glycinate
(AA-1)
pKa1=2.35 pKa
2=9.78
Implementing in-line dilution requiresIn-depth understand of buffer chemistrySolubility and stability of buffer concentratesEffect of temperature on pH and mS/cmAppropriate equipment for in-line dilution
15/04/2005 / 17
Protein A Equilibration Buffer Chemistry
5 10 15 20 25 307.85
7.90
7.95
8.00
8.05
8.10
8.15
8.20
8.25
8.30
pH (-
)
Temperature (°C)
16
18
20
22
24
26
Con
duct
ivity
(mS
/cm
)
0 2 4 6 8 107.85
7.90
7.95
8.00
8.05
8.10
8.15
8.20
8.25
8.30
pH (-
) Concentrate Strength (-)
0
20
40
60
80
100
120
140
160
Con
duct
ivity
(mS
/cm
)
Effect of temperature on at-strength buffer pH & mS/cm
Static dilution of buffer concentrates
Establish process tolerance for pH and mS/cm (as wide as possible)
Check ability to control concentrate and WFI temperature
Ensure buffer concentrate is soluble at required strength
Check Salt Strength at pH for compatibility with MOC’s
15/04/2005 / 18
0 20 40 60 80 100 1200
102030405060708090
100
% B
Volumetric Flowrate (L/h)
10x Dilution
5x Dilution
Demonstrating potential for in-line dilution
15/04/2005 / 19
0 20 40 60 80 100 1200
102030405060708090
100
% B
Volumetric Flowrate (L/h)
10x Dilution
5x Dilution
10x Concentrate dilution at 20L/h
CIR101 CIR102 AIR121pH FIR141 TIR101 SetMark
0
50
100
150
mS/cm
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
pH
0.0 2.0 4.0 6.0 8.0 10.0 min
Wat
er ri
nse
1x b
asel
ine
10x_
conc
entr
ate
Feed
back
ena
bled
10x concentrate: 500mM Gly-Gly, 2500mM NaCl, 8.03pH, 166.8 mS/cm
Controller set point = 15%B for 10x dilution to 25.1mS/cm
15/04/2005 / 20
10x Concentrate dilution at 120L/h
0 20 40 60 80 100 1200
102030405060708090
100
% B
Volumetric Flowrate (L/h)
10x Dilution
5x Dilution
CIR101 CIR102 AIR121pH FIR141 TIR101 SetMark
0
50
100
150
mS/cm
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
pH
0.0 2.0 4.0 6.0 8.0 10.0 min
Wat
er ri
nse
1x b
asel
ine
10x_
conc
entr
ate
Feed
back
ena
bled
21.0
22.0
23.0
24.0
25.0
26.0
27.0
21.0
22.0
23.0
24.0
25.0
26.0
27.0
6.0 7.0 8.0 9.0 10.0 min
Feed
back
ena
bled
15/04/2005 / 21
Importance of in-line dilution for addressing demands of high titre batches.
Careful selection of buffers during early phase development can alleviate serious headaches at scale later
Select buffers (especially equilibration/wash) that can be prepared in concentrate form
Design capability for in-line dilution into chromatography and ultrafiltration rigs
Possible to obtain a 30-40% reduction in total buffer prep and hold requirements for equilibration buffers alone
15/04/2005 / 22
Process Development Case Study 2
Throughput optimisation-Protein A capture
Recent advances in Protein A matrix designHigh throughput matricesHigh capacity matricesAlkali stable options
Explore relationship between titre increase and matrix selection
Examine impact of column diameter on process design and operation
15/04/2005 / 23
Effect of fermentation titre on column capacity
0 10 20 30 40 500
20
40
60
80
100
IgG loaded (mg/mL.matrix)
PnA Sepharose, 5g/L
Bre
akth
roug
h (C
/Co)
%
PnA Sepharose, 0.5g/L 10x increase in fermentation titre
83% increase in binding capacity under identical conditions
Despite increased binding capacity, compressibility limits attainable throughput and scale-up potential
0 100 200 300 400 500 600 7000
5
10
15
20
25
30
35
40
Pres
sure
Dro
p (p
si)
Linear velocity (cm/h)
1.6cm 2.6cm 5.0cm 10cm 20cm 28cm 40cm
ColumnDiameter
140cm
15/04/2005 / 24
Addressing chromatographic throughput
Incompressible matrices offer benefit of higher throughput.
Essential for high volume fermenters
Breakthrough curves generated at 450cm/h
(3x faster than Sepharose maximum )
23% increase in binding capacity at 5% C/Co under identical conditions.
0 10 20 30 40 500
20
40
60
80
100 MabSelect, 0.5g/L
Bre
akth
roug
h (C
/Co)
%
IgG loaded (mg/mL.matrix)
MabSelect, 5g/L
15/04/2005 / 25
Importance of matrix selection on process throughput at different titres
0.5g/L Titre 5g/L Titre
Rmp Protein ASepharose FF
MabSelect
ConclusionHigh throughput matrices can deliver 50% improvement in throughput at high fermentation titres
2000L Reactor
15/04/2005 / 26
Influence of column diameter on batch and campaign costs.
2000L reactor at 5g/LMabSelect for initial capture
“Small” PnA Column
“Large” PnA Column
No difference in campaigncost over 15 batches
15/04/2005 / 27
Factors to consider when selecting a column diameter
Range of fermentation titres across all products in portfolio (Flexibility)
A wide range in titre drives higher utilisation of narrower column diameters
Campaign length (number of batches)longer campaigns may favour large diameter columns
RiskBioburden contamination, operating close to re-use limits
Capital equipment requirementsmore chromatography skids, larger elution tanks)
Facility design, operation, logisticsfloor space-processing/storage, packing-unpacking
15/04/2005 / 28
Adressing capacity challenges with larger columns
Small diameter columns
Lower capital costsColumns and skids
Lower one-off batch costs
Increased flexibility across range of titres
Potential issues with high utilisation rates of equipment
MaintenanceColumn Re-packing
Large diameter columns
Higher capital costsColumns (stainless?)Skids
Extremely high first batch and replacement PnA costs
Large elution volumes per cycle
High exposure to risk through contamination (e.g. bio-burden) or other process failure
15/04/2005 / 29
Stay abreast of latest vendor offerings
1991 Protein A Seph 4FF
1996 rProtein A Seph 4FF
2000 rmp Protein A Seph 4FF
2001 MabSelect
2005….MabSelect SuRe, Xtra
Amersham Matrix launches.
E D A B C X MSs
Immunoglobulin binding domains
Gly29Ala mutation
Z domain
15/04/2005 / 30
Higher through, higher capacity, alkali stability
0 10 20 30 400
20
40
60
80
100 rmpPnA Seph 4 FF
Bre
akth
roug
h (C
/Co)
%
Antibody Loaded (mg/mL)
Mabselect
Prototype SuRe
Rmp Protein A 4 FFXK16, 15cm Ho, 150cm/h, 6min RT5% C/Co = 26g/LOp.DBC = 20.8g/L
MabSelectTC5, 20cm Ho, 500cm/h, 2.4min RT5% C/Co = 19g/LOp.DBC = 15.2/L
MabSelect SuReTC5, 20cm Ho, 500cm/h, 2.4min RT5% C/Co = 26g/LOp.DBC = 20.8g/L
15/04/2005 / 31
Strategies for Chromatography Process Development
Ensure process development teams understand manufacturing requirements:
High throughput per batch?Low batch costs?
Operate closer to 1% Breakthrough to minimise cycling requirements
Operate as close to matrix throughput limits as manufacturing capabilities permit (minimise residence time)
Employ latest matrix technologies
Ensure processes have detailed cost models to justify/defend process decisions
15/04/2005 / 32
Individual Reactor Volume established at 20,000L
LONZA BiologicsLSBO Facility
3x20,000L Reactors
Increasing to 4 Reactors in 2006
Purified through a Single Purification train
15/04/2005 / 33
Do ultra-large columns adequately meet high titre challenge?
LSBO Facility designed to handle high-titre batches
Protein A Capture in either a 1.4 or 2.0 m diameter Chromatography column.
Chromatographic capture of 20,000L at 5g/L feasible given facility design basis
15/04/2005 / 34
Process Development Case Study 3
Ultrafiltration Optimisation
Early Phase fast track development may not allow sufficient time to optimise UF operations
Diafiltration conditions may be selected based onPrior experience with antibody class/pIPrior experience with particular cassette type/vendorManufacturing equipment capabilitiesAntibody stability
UF optimisation can substantially improve purification process performance
15/04/2005 / 35
Ultrafiltration Optimisation
TMP
Flux
-J
Low QMed QHigh Q
cTMP
Bulk Flow (Cb)
Boundary Layer
Gel Layer (Cg)Membrane
Increasing Concentration
Cb
Cg
Crossflow
eC
C gdf =
ln C CgFl
ux -
J
15/04/2005 / 36
Results from an Ultrafiltration optimisation
Membrane Un-Optimised Optimised
Gel Concentration (Cg) (g.L-1) 44.6 72.1
Optimum Process Conc (Cg/e) (g.L-1) 16.4 26.5
Flux at Cg/e (L.m-2.h-1) 31.1 59.3
Diafiltration Volume (L) 2,332 1,443
5 x Diafiltration Volume (L) 11,660 7,215
Concentration Time (h) 2 h 32 min 1 h 22 min
Diafiltration Time (h) 9h 22 min 3h 2 min
Total Process Time (h) 11 h 54 min 4 h 24 min
Un-optimised
Pin = 20 psig
Pout = 5 psig
Cross flow = 1x
Optimised
Pin = 35 psig
Pout = 12 psig
Cross flow = 1.33x
Membrane Area = 40 m2
Initial Volume = 7,300 L
Initial concentration = 5.24 g/L, concentrated to membrane Cg
15/04/2005 / 37
0 10 20 30 40 50 60 70 80 90 100 1100
2
4
6
8
10
12
14
Tota
l Pro
cess
Tim
e (h
)
Product Concentration at DF (g.L-1)
Omega Biomax Hydrosart Alpha 5 x DF Volume
0
2k
4k
6k
8k
10k
12k
14k
16k
18k
20k
Dia
filtra
te V
olum
e (L
)
Ultrafiltration OptimisationInfluence of Cassette type and vendor
KeyPolyethersulfone Vendor 1Polyethersulfone Vendor 2Regen. Cellulose Vendor 2Regen. Cellulose Vendor 3
Wide variation in optimised performance across different UF cassettes, especially at extremities of protein concentration
Model: 40kg batch
Membrane Area = 40 m2
Initial Volume = 7,300 L
Initial concentration = 5.24 g/L, concentrated to Cg, 5 diavolumes.
15/04/2005 / 39
Ultrafiltration Optimisation
Optimisation of UF operations can deliver reduced processing times and considerable reduction in buffer volumes
Simple ultrafiltration optimisation can be performed in a single day with tremendous benefits
Avoid mid-range operating conditions-lead to un-optimised processes
Product stability at elevated concentrations should be assessed with appropriate stability studies
15/04/2005 / 40
Summary
Maximise utilisation of existing assets for as long as possible“Optimise” performance of unit operations early in development life cycle
Employ high throughput chromatography matrices
Select buffers with in-line dilution in mind
Strive for purification at elevated protein concentration wherever possible
However above strategy may have only a limited lifespan
Parallel track with investment into novel and conventional technologies