Use of GXII Platform in HT Screening for Mammalian ... - PerkinElmer
Use of GXII Platform in HT Screening for Mammalian ... - PerkinElmer
Use of GXII Platform in HT Screening for Mammalian ... - PerkinElmer
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Perk<strong>in</strong>Elmer <strong>Use</strong>r Meet<strong>in</strong>g – 12 June 2012<br />
<strong>Use</strong> <strong>of</strong> <strong>GXII</strong> <strong>Plat<strong>for</strong>m</strong> <strong>in</strong> <strong>HT</strong> Screen<strong>in</strong>g <strong>for</strong><br />
<strong>Mammalian</strong> Cell Culture Prote<strong>in</strong> Development<br />
Imtiaz Alam, Head <strong>of</strong> Process Analytics / R&D Operational Excellence Specialist<br />
Lonza Biologics plc / 2012
Scope <strong>of</strong> Presentation<br />
• Bio-pharmaceutical Products<br />
• Role <strong>of</strong> Process Analytics<br />
• Why Speed Up Process Analytics Support <strong>for</strong><br />
Product Development?<br />
• Challenges to Process Analytics<br />
• <strong>GXII</strong> / Sciclone as Part <strong>of</strong> the <strong>HT</strong> Analytics<br />
Screen<strong>in</strong>g <strong>Plat<strong>for</strong>m</strong><br />
• Summary<br />
© Lonza Biologics plc 2012 slide 2
Bio-pharmaceutical Products<br />
• Therapeutic prote<strong>in</strong> production by recomb<strong>in</strong>ant–DNA<br />
technology.<br />
• Genes encod<strong>in</strong>g the prote<strong>in</strong> are transfected <strong>in</strong>to a target<br />
cell l<strong>in</strong>e.<br />
• Transfected cells are generally cloned and adapted to<br />
secrete large amounts <strong>of</strong> product dur<strong>in</strong>g a fermentation<br />
culture.<br />
• The primary, secondary and tertiary structure <strong>of</strong> the<br />
prote<strong>in</strong> product is controlled by the cell.<br />
• The product is purified from the cell culture medium.<br />
© Lonza Biologics plc 2012 slide 3
Process Analytics<br />
• Requirement <strong>for</strong> High Throughput Analytical Test<strong>in</strong>g / Screen<strong>in</strong>g <strong>for</strong><br />
Product Characteristics exist through the whole product lifecycle:<br />
• Cell l<strong>in</strong>e development<br />
• Process development<br />
• Fermentation<br />
• Purification<br />
• Process optimization<br />
• Process scale up<br />
• Process validation<br />
• Process transfer<br />
• F<strong>in</strong>al product test<strong>in</strong>g<br />
• Reference standard characterization<br />
• Biochemical comparability<br />
• Stability studies<br />
• Post-market surveillance<br />
© Lonza Biologics plc 2012 slide 4
Reduce Timel<strong>in</strong>es <strong>for</strong> Recomb<strong>in</strong>ant<br />
Prote<strong>in</strong> Production<br />
• Strategy to reduce the timel<strong>in</strong>e <strong>for</strong> recomb<strong>in</strong>ant prote<strong>in</strong><br />
by 50%<br />
• Timel<strong>in</strong>e sav<strong>in</strong>gs from us<strong>in</strong>g advance host cell l<strong>in</strong>e<br />
(CHOK1SV GS-KO)<br />
• Automation <strong>of</strong> manual cell culture development processes<br />
• Need to screen <strong>for</strong> product characteristic earlier <strong>in</strong> the process<br />
• Implementation <strong>of</strong> m<strong>in</strong>iaturized bioreactors (Amber System)<br />
• Shift to 'product by design' which requires characterization <strong>of</strong><br />
the product from large numbers <strong>of</strong> cell l<strong>in</strong>es<br />
© Lonza Biologics plc 2012 slide 5
Colony Identification, Sampl<strong>in</strong>g and Pick<strong>in</strong>g<br />
– Islands <strong>of</strong> Automation<br />
ICCMS<br />
Hamilton Star<br />
Cytomat<br />
<strong>in</strong>cubator<br />
sample<br />
list<br />
clon<strong>in</strong>g<br />
plates<br />
Cytomat<br />
<strong>in</strong>cubator<br />
CSI<br />
samples<br />
pick list<br />
Off-l<strong>in</strong>e productivity<br />
assessment<br />
(product specific)<br />
© Lonza Biologics plc 2012 slide 6
Automated M<strong>in</strong>iature Bioreactors<br />
– Islands <strong>of</strong> Automation<br />
• Automated Bioreactors <strong>Plat<strong>for</strong>m</strong><br />
• 10 samples to 100`s<br />
• Product Quality Assessment<br />
• PhyTip purification<br />
• Prote<strong>in</strong> purity<br />
• MW determ<strong>in</strong>ation<br />
• IEF<br />
• Glycan<br />
• Activity<br />
15ml volume<br />
TAP ambr system<br />
© Lonza Biologics plc 2012 slide 7
Reduce Timel<strong>in</strong>es <strong>for</strong> Recomb<strong>in</strong>ant<br />
Prote<strong>in</strong> Production<br />
• Microscale purification development (res<strong>in</strong> and condition<br />
scout<strong>in</strong>g)<br />
• DoE <strong>for</strong> process optimization <strong>in</strong> cell culture, fermentation and<br />
10L and m<strong>in</strong>iature bioreactors<br />
• Prevent ULD and high team stress due to workload<br />
© Lonza Biologics plc 2012 slide 8
Purification Optimisation Robot<br />
– Islands <strong>of</strong> Automation<br />
• Automated Purification<br />
<strong>Plat<strong>for</strong>m</strong><br />
• Atoll Robo Columns<br />
• Onl<strong>in</strong>e HCP ELISA<br />
• Buffer prep<br />
• UV Reader<br />
• Onl<strong>in</strong>e SEC HPLC<br />
• Product Quality Assessment<br />
• Prote<strong>in</strong> purity<br />
• IEF<br />
• Glycan<br />
© Lonza Biologics plc 2012 slide 9
12<br />
10<br />
Demand Pattern, Probability <strong>of</strong><br />
Turnaround, Schedul<strong>in</strong>g, Skills Sets<br />
Sample Purification - Probability <strong>of</strong> Insuficient<br />
Capacity <strong>in</strong> Any Given Week<br />
8<br />
6<br />
4<br />
2<br />
0<br />
1<br />
2<br />
3<br />
4<br />
5<br />
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50<br />
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52<br />
53<br />
Data<br />
Sum <strong>of</strong> DRIPPY Sum <strong>of</strong> Purifn Sum <strong>of</strong> MEA<br />
Wk_No<br />
50%<br />
40%<br />
30%<br />
20%<br />
10%<br />
0%<br />
Probability <strong>of</strong> Insufficient<br />
Capacity <strong>in</strong> any given week<br />
4 5 6 7 8 9<br />
Assay Capacity (MEA)<br />
2006<br />
2007<br />
2008<br />
© Lonza Biologics plc 2012 slide 10
Applications <strong>of</strong> <strong>HT</strong> Screen<strong>in</strong>g<br />
Antibody<br />
productivity,<br />
assembly and titer<br />
Medium selection <strong>for</strong><br />
optimal antibody<br />
production<br />
Purification<br />
development,<br />
optimal conditions<br />
<strong>for</strong> b<strong>in</strong>d<strong>in</strong>g, elution,<br />
impurities, yield<br />
Electronic Tomography<br />
“Lonza B72.3 Antibody”<br />
Lab scale fermentation<br />
process, titer,<br />
monomer/aggregation,<br />
purity, glycosylation and<br />
fragments<br />
© Lonza Biologics plc 2012 slide 11
Challenges to Process Analytics:<br />
Automation and Throughput<br />
Requirement<br />
Rapid turnaround<br />
Sample demand<br />
Improvements to the Process<br />
From 5 - 10 days to
Tools <strong>for</strong> <strong>HT</strong> Product Characteristic Screen<strong>in</strong>g<br />
Tests Standard Methods <strong>HT</strong> Screen<strong>in</strong>g Tools<br />
Analytical scale<br />
sample purification<br />
PhyTips / Semi automated<br />
(10`s <strong>of</strong> samples)<br />
<strong>GXII</strong> / Sciclone <strong>Plat<strong>for</strong>m</strong> &<br />
PhyTips (48 to 96 samples)<br />
% Purity SDS PAGE (10 samples) <strong>GXII</strong> / Sciclone <strong>Plat<strong>for</strong>m</strong><br />
(100 to 200 samples)<br />
Charge analysis - Agrose Gel, CZE (10 to 24<br />
samples)<br />
To review use <strong>of</strong> <strong>GXII</strong><br />
Glycan pr<strong>of</strong>ile<br />
Aggregation<br />
MALDI TOF MS, HPLC &<br />
UPLC (4 to 8 samples)<br />
HPLC & UPLC<br />
(24 samples)<br />
<strong>GXII</strong> / Sciclone <strong>Plat<strong>for</strong>m</strong><br />
UPLC<br />
<strong>GXII</strong> / Sciclone <strong>Plat<strong>for</strong>m</strong><br />
© Lonza Biologics plc 2012 slide 13
<strong>HT</strong> Analytics <strong>Plat<strong>for</strong>m</strong><br />
• Rapid turnaround <strong>of</strong> analytical support is essential<br />
• Sample preparation<br />
• Analytical test<strong>in</strong>g<br />
• Data analysis and report<strong>in</strong>g<br />
• To screen prote<strong>in</strong>s <strong>for</strong> quality and characteristics attributes<br />
<strong>in</strong> a <strong>HT</strong> <strong>for</strong>mat <strong>for</strong><br />
• Timel<strong>in</strong>e reduction<br />
• Optimal cell l<strong>in</strong>e/ process parameters<br />
• Lonza Process Analytics has <strong>in</strong>itiated a phased approach<br />
to <strong>in</strong>tegrate <strong>HT</strong> technology<br />
© Lonza Biologics plc 2012 slide 14
<strong>GXII</strong> / Sciclone as Part <strong>of</strong> the<br />
<strong>HT</strong> Analytical Screen<strong>in</strong>g <strong>Plat<strong>for</strong>m</strong><br />
Phase I Phase II Phase III<br />
CCS Purification –<br />
PhyTips<br />
SDS, Glycan Screen<strong>in</strong>g<br />
Prote<strong>in</strong> Concentration (UV)<br />
Sample Preparation, IEF capability,<br />
Increased throughput (In plann<strong>in</strong>g)<br />
Aggregation, full range Glycan, Sialic Acid<br />
K<strong>in</strong>etics / Activity analysis (In plann<strong>in</strong>g)<br />
© Lonza Biologics plc 2012 slide 15
Phase I – URS Overview (25 page document)<br />
• The <strong>HT</strong> Cell Culture Analytics <strong>Plat<strong>for</strong>m</strong><br />
schematic:<br />
Liquid Handler Robot<br />
Cell Culture Supernatant Sample<br />
(from Cell Culture Development)<br />
UV Spectrophotometer<br />
Chilled deck<br />
position<br />
Sample purification<br />
Read Absorbance<br />
at A280<br />
• Automated steps (walk away 24/7 process)<br />
• 1: Sample purification from CCS <strong>in</strong>to<br />
partially purified material – PhyTips<br />
• 2: Concentration determ<strong>in</strong>ation <strong>of</strong> eluate<br />
(UV spec)<br />
• 3: Automated normalization <strong>of</strong> concentrations<br />
and production <strong>of</strong> sample plates <strong>for</strong> purity &<br />
glycan analysis<br />
• 4: Sample preparation <strong>for</strong> prote<strong>in</strong> purity and<br />
glycan screen<strong>in</strong>g<br />
• 5: Analysis and electronic data transfer<br />
Heated deck<br />
position<br />
Concentration normalisation (dilution)<br />
Creation <strong>of</strong> daughter plates (samples <strong>for</strong> analysis)<br />
Sample preparation <strong>for</strong> CE analysis<br />
Capillary Electrophoresis Analytical Instrument<br />
Analysis<br />
Data export as delimited text file (.csv) or <strong>in</strong>to<br />
external CDS (Empower)<br />
Physical manual movement<br />
Physical automated movement<br />
Data transfer<br />
Process step<br />
Included function<br />
Determ<strong>in</strong>e<br />
Concentration<br />
Create job list <strong>for</strong><br />
liquid handler<br />
Plate map report (<strong>in</strong>clud<strong>in</strong>g<br />
concentrations)<br />
Standalone purified samples <strong>for</strong><br />
preparation <strong>for</strong> CE analysis<br />
Standalone prepared samples <strong>for</strong> analysis<br />
© Lonza Biologics plc 2012 slide 16
Phase I – PA <strong>HT</strong> <strong>Plat<strong>for</strong>m</strong> on the Bench<br />
Integrated plat<strong>for</strong>m<br />
• <strong>GXII</strong> (prote<strong>in</strong> purity +<br />
MW and glycan<br />
analysis)<br />
• Sciclone Liquid Handler<br />
(Integrated <strong>in</strong> UV spec)<br />
• Twister II arm, <strong>for</strong><br />
additional plate storage<br />
and plate movement to<br />
all components <strong>of</strong> the<br />
plat<strong>for</strong>m<br />
© Lonza Biologics plc 2012 slide 17
Application –<br />
Prote<strong>in</strong> Purification Us<strong>in</strong>g PhyTips<br />
• Application to use Sciclone Liquid Handler to automate<br />
prote<strong>in</strong> purification <strong>in</strong> PhyTips<br />
© Lonza Biologics plc 2012 slide 18
Rapid Product Quality<br />
SDS PAGE Traditional method<br />
1.5 days<br />
15 m<strong>in</strong> 2 to 3 hours 2 to 3 hours 1 hour<br />
Prep Gel Run Gel Sta<strong>in</strong>/Desta<strong>in</strong> Scan/Analyze<br />
15 m<strong>in</strong> 25 m<strong>in</strong><br />
Prep Chip<br />
Run Chip<br />
X 10 sample capacity<br />
Analysis<br />
1 hour<br />
750<br />
650<br />
550<br />
Fluorescence<br />
450<br />
350<br />
250<br />
150<br />
50<br />
-50<br />
17 22 27 32 37 42<br />
Time (seconds)<br />
© Lonza Biologics plc 2012 slide 19
L<strong>in</strong>k<strong>in</strong>g Islands <strong>of</strong> Automation to Form an<br />
“Archipelago”<br />
Productivity<br />
assessment<br />
1<br />
Transfect<br />
Generate<br />
stable<br />
pools<br />
Clon<strong>in</strong>g by<br />
FACS<br />
(1 cell/well)<br />
Automated<br />
colony<br />
identification<br />
(ICCMS)<br />
Automated<br />
colony<br />
sampl<strong>in</strong>g<br />
Automated<br />
Colony<br />
pick<strong>in</strong>g<br />
Screen<strong>in</strong>g, DoE,<br />
optimisation<br />
Bioreactor studies<br />
etc.<br />
Stability <strong>for</strong><br />
manufactur<strong>in</strong>g<br />
WEEK 17*<br />
choose<br />
lead cell<br />
l<strong>in</strong>e<br />
3<br />
Fed-batch productivity & PQ, 9 cell l<strong>in</strong>es<br />
Fed-batch<br />
productivity<br />
assessment<br />
© Lonza Biologics plc 2012 slide 20<br />
2<br />
WEEK ~9*<br />
choose<br />
candidate<br />
cell l<strong>in</strong>es<br />
Shak<strong>in</strong>g 96-DWP<br />
Rapid Product Quality<br />
• Early <strong>in</strong>tegrity and identity screen<strong>in</strong>g<br />
• Advances <strong>in</strong> “Lab-on-a-Chip” technology to replace traditional<br />
gel based SDS electrophoresis<br />
• Rapid capillary based separation<br />
•
Case Study: Prote<strong>in</strong> Fragment production<br />
% Integrity<br />
100.0<br />
90.0<br />
80.0<br />
70.0<br />
60.0<br />
50.0<br />
40.0<br />
30.0<br />
20.0<br />
10.0<br />
0.0<br />
0.0 1.0 2.0 3.0 4.0 5.0<br />
Time (Hours)<br />
• Manufactur<strong>in</strong>g process<br />
required rapid antibody<br />
<strong>in</strong>tegrity test<strong>in</strong>g<br />
• Not possible by standard<br />
techniques (SDS PAGE)<br />
• Allowed decision mak<strong>in</strong>g<br />
driven by data to obta<strong>in</strong><br />
optimum yield<br />
Aggregate (>200 kDa)<br />
Prote<strong>in</strong> XS1 (160 to 140 kDa)<br />
Product (106 kDa)<br />
Fragment A (100 to 92 kDa)<br />
Fragments B (90 to 30 kDa)<br />
© Lonza Biologics plc 2012 slide 22
Glycan Pr<strong>of</strong>il<strong>in</strong>g Workflow<br />
• A microchip-CE method has been developed <strong>for</strong> pr<strong>of</strong>il<strong>in</strong>g<br />
N-l<strong>in</strong>ked glycan's<br />
• The five major glycan peaks are easily resolved <strong>in</strong> less<br />
than 45 seconds per sample<br />
• Assay precision is
Why Screen <strong>for</strong> Glycosylation Dur<strong>in</strong>g<br />
Early Process Development?<br />
Glycans regulate:<br />
•Biological activity<br />
•Half-life<br />
For MAbs:<br />
Fc region Oligos <strong>in</strong>teract<br />
with other immune<br />
system prote<strong>in</strong>s, mak<strong>in</strong>g<br />
them essential <strong>for</strong><br />
antibody function.<br />
© Lonza Biologics plc 2012 slide 24
Case Study: Glycan by MS/HPLC and <strong>GXII</strong><br />
• 200 Cell l<strong>in</strong>e DoE Screen<strong>in</strong>g Study<br />
• Relative % changes <strong>in</strong> ma<strong>in</strong> Glycan<br />
structures<br />
• >40 Bioreactors<br />
Issues MS / HPLC / UPLC <strong>GXII</strong> <strong>HT</strong> <strong>Plat<strong>for</strong>m</strong><br />
Process Risk<br />
Discussion between 3 sites to plan<br />
test<strong>in</strong>g slots, hardware & multiple<br />
Scientists<br />
1 Scientist<br />
Turnaround 6 to 8 weeks
Summary<br />
• “Islands <strong>of</strong> Automation” approach is ideal to ensure process can be<br />
automated <strong>for</strong> a specific function / required capacity / makes<br />
automation achievable<br />
• <strong>Use</strong> exist<strong>in</strong>g manual/data l<strong>in</strong>ks to build the ““Archipelago” <strong>for</strong> full<br />
scale <strong>in</strong>tegration<br />
• The <strong>GXII</strong> M<strong>in</strong>iaturised <strong>HT</strong> multi-analyte plat<strong>for</strong>m is essential to<br />
ensure analytics is no longer a limitation to timel<strong>in</strong>e reduction and<br />
automation <strong>of</strong> therapeutic prote<strong>in</strong> development<br />
© Lonza Biologics plc 2012 slide 26
Acknowledgments<br />
• Monica Barredo<br />
• Lewis Higg<strong>in</strong>s<br />
• Gareth Meek<br />
• Ramsha Qureshi<br />
• Adrian Ha<strong>in</strong>es (Cell Culture)<br />
• Andy Racher (Cell Culture)<br />
Perk<strong>in</strong> Elmer Team<br />
• Darren Stubbs<br />
• Kathryn Hawkesworth<br />
• Mark Rowe<br />
• Richard Sawk<strong>in</strong>s<br />
© Lonza Biologics plc 2012 slide 27
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