ProPac Elite WCX, 5 μm Particle, for Fast, High Resolution Protein and mab Analysis. July 2018

Transcription

1 ProPac Elite WCX, 5 μm Particle, for Fast, High Resolution Protein and mab Analysis July 218

2 ProPac Elite WCX Acidic Variants Basic Variants 5µm Weak Cation Exchange Column Chemistry based on Thermo Scientific ProPac WCX-1 Main Application: Charge Variant Analysis Separating a protein based on the on accessible surface charge. Deamidation, lysine truncation, glycosylation are all terms customers will use. The analysis of charged variants is a regulatory requirement for bio-therapeutic proteins Main Market: Biopharmaceutical/ Pharmaceutical and University/Academic 2

3 Why was this Product Created 1998 ProPac WCX-1, 1 µm ProPac SCX-1, 1 µm 21 ProPac SCX-2, 1 µm MAbPac SCX-1, 1 µm 212 MAbPac SCX-1, 3 µm 218 ProPac Elite WCX, 5 µm 211 MAbPac SCX-1, 5 µm 213 MAbPac SCX-1 RS, 5 µm CX-1 ph Gradient Buffer More reproducible resin/less requirement for lot selection for customers Speed/ higher throughput methods Brand recognition A high number of journal articles focus on WCX/ not all customers want a SCX chemistry The name ProPac still has a draw for people not running monoclonal antibodies, just therapeutic proteins 3

4 2 Year History 218 Thermo Fisher Scientific Inc. All rights reserved. Google is a registered trademark of Google LLC. HERCEPTIN is a trademark of GENENTECH, INC. Apple imac is a trademark of Apple Inc. All other trademarks are the property of Thermo Fisher Scientific. This information is presented as an example of the capabilities of Thermo Fisher Scientific products. It is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. 4

5 Marketing Campaign Teaser Campaign- one week before launch Launch Date- July 29 th Posters/Trade Show 3 posters on ProPac Elite Follow up s Banners At launch -Specification Sheet -Three Application Notes Following Launch -Brochure -Additional Application Note -Technical Note Webinar in September focusing on alt. uses -comparison with ice -use of MS friendly buffers 5

6 How to Position Against Existing Portfolio ProPac Elite WCX builds on the legacy of the ProPac WCX-1. Optimized. ProPac WCX-1 was introduced 2 years ago. This column uses similar chemistry, but enhancements were made with the customer experience in mind Lot-to-lot performance High resolution separations Option for fast method development 4x15mm ProPac Elite WCX offers same resolution as 4x25mm ProPac WCX-1 in less time 2x5mm format ProPac Elite WCX offers separations in under 1 minutes for high throughput labs ProPac WCX-1 is NOT being obsoleted This is not in competition to the MAbPac SCX-1, nor a replacement. It is a complementary product in a complete portfolio of solutions 6

7 ProPac Elite WCX Column Chemistry 1 Smaller Particle Size 2 Improved Column Chemistry Improved lot-to-lot consistency 3 Similar Selectivity ProPac WCX-1 ProPac Elite WCX 7

8 Reproducibility: Lot-to-Lot Lot 1 Lot 2 Lot 3 Lot 4 Retention time mab Cyto. C RNase A Lot Lot Lot Selectivity RT (3-2)/(2-1) Lot 5 Lot 6 Lot 7 Lot 8 Lot 9 Lot 1 Lot Lot Lot Lot Lot Lot Lot % RSD

9 Reproducibility: Injection-to-Injection % Area mab % Area Variants 7 Main peak Calculation of % Peak Area mab Acidic Basic Overlay of 1 consecutive injections Acidic Basic Injection 9

10 Two Methods for Protein/mAb Separation on IEX Columns Salt Gradient ph Gradient Salt disrupts protein-solid phase interactions Change in protein charge disrupts protein-solid phase interactions Most widely used method Relatively simple to make the buffer Buffer design (ph, salt concentration) can optimize the separation Can predict elution profile with pi value Lower salt concentration in collected fractions CX-1 ph Gradient Buffers provide a linear ph gradient 1

11 ProPac Elite WCX and CX-1 ph Gradient Buffers ph Gradient Change in protein charge disrupts protein-solid phase interactions Can predict elution profile with pi value CX-1 ph Gradient Buffer A (ph 5.6) CX-1 ph Gradient Buffer B (ph 1.2) Lower salt concentration in collected fractions CX-1 ph Gradient Buffers provide a linear ph gradient 1X Concentration 11

12 Protein ph Gradient Elution Mechanism on CEX ph 11 ph Resin ph

13 Protein ph Gradient Elution Mechanism on CEX ph 11 Negative 1 Isoelectric point (pi) Neutral - - ph Positive ph

14 Protein ph Gradient Elution Mechanism on CEX ph 11 Negative 1 Isoelectric point (pi) Neutral - - ph Positive ph

15 Protein ph Gradient Elution Mechanism on CEX ph

16 There is Gradient Delay on the WCX Stationary Phase ph Required equilibration times are listed in the manual SCX WCX

17 ph Gradient Analysis of Pharmaceutical mabs 6 Rituximab 33 Trastuzumab Column: ProPac Elite WCX, 5 µm Format: 4 15 mm Mobile phase A: CX-1 ph Gradient Buffer A Mobile phase B: CX-1 ph Gradient Buffer B Gradient: 25% B change in 15 minutes Flow rate:.8 ml/min Temperature: 3 ºC Detection: UV (28 nm) Inj. volume: 4 µl (5 mg/ml) 4 Infliximab 1 Secukinumab

18 Salt Gradient Method Salt Gradient Salt disrupts protein-solid phase interactions Most widely used method Relatively simple to make the buffer Buffer design (ph, salt concentration) can optimize the separation A 2 mm MES, ph 6.5 B 2 mm MES, ph M NaCl 18

19 Compatible Buffers and Salts Recommended Buffers Good s Buffers pka (25 ºC) ph Range MES MOPS HEPES TAPS CAPSO Recommended Salts Sodium chloride (NaCl) Typically 1 M or.5 M concentration. Typically 2 to 5 mm concentration Sodium phosphate? Potassium phosphate? This buffer was recommended in the original documentation for the ProPac WCX-1. We do NOT recommend customers to adapt methods using these buffers to the new column. It is NOT an effective buffer 19

20 Buffer ph Effect 14 MES ph Column: ProPac Elite WCX 5 µm Format: 4 15 mm Eluents: See chromatogram for specific buffer and ph A: 2 mm Buffer B: 2 mm Buffer +.5 M NaCl 12 MES ph Gradient: %A %B MOPS ph Flow Rate: 1. ml/min Inj. Vol: 3 µl Temp: 3 o C Detection: UV, 28 nm Sample: Rituximab, 5 mg/ml Peak Label: Retention time (min) 2

21 Salt Gradient Analysis of Pharmaceutical mabs 6 Rituximab %B (loading): 8 %B (initial): 13 %B (final): 22 2 Trastuzumab %B (loading): 9 %B (initial): 12 %B (final): 2 Column: ProPac Elite WCX, 5 µm Format: 4 15 mm Mobile phase A: 2 mm MES, ph 6.5 Mobile phase B: 2 mm MES,.5 M NaCl, ph 6.5 Gradient: Loading:..8 min Gradient: min Wash: min Flow rate: 1. ml/min Temperature: 3 ºC Detection: UV (28 nm) Inj. volume: 3 µl (5 mg/ml) 23 Infliximab 57 Secukinumab %B (loading): 4 %B (initial): 8 %B (final): 18 %B (loading): 5 %B (initial): 1 %B (final):

22 Ruggedness: Stable Over 5 Runs 45 Salt Gradient 8 ph Gradient Flow Rate: 1 ml/min Flow Rate:.8 ml/min Run 5 Run 5 Run 1 Run

23 Ruggedness: Stable Over 5 Runs Pressure (bar) Pressure (bar) 5 Salt Gradient 5 ph Gradient Flow Rate: 1 ml/min Flow Rate:.8 ml/min 4 4 Run 5 3 Run 1 3 Run 5 Run

24 Warning to Ensure Column Ruggedness Manual and QuickStart CAUTION NEVER use pure deionized water on the column as this will result in irreversible column damage! WHY? Column Minimum 2mM NaCl required 24

25 Loading Capacity Measured Using a mab Sample PWHH Main Peak (min) PWHH Main Peak (min) 1,6 Salt Gradient 2,5 ph Gradient mg/ml column volume (CV) mg/ml column volume (CV) µg mab Loaded µg mab Loaded 25

26 Loading Capacity Table Column Format Loading Capacity (Salt Gradient Method; µg) Loading Capacity (ph Gradient Method; µg) 2x5 mm x15 mm x25 mm x5 mm x15 mm x25 mm Note: This is based on a IgG1 mab (biosimilar of trastuzumab). Exact loading capacity will depend on the protein. 26

27 Column Format Guide 16 4x5 mm 5 min Column: ProPac Elite WCX, 5 µm Format: See chromatogram Mobile phase A: 2 mm MES, ph 6.5 Mobile phase B: 2 mm MES +.5 M NaCl, ph x15 mm 15 min Gradient: Flow rate:.6 ml/min Temperature: 3 ºC Detection: UV (28 nm) Loading 9% B 12-22% B; see chromatogram for time of gradient 25 4x25 mm 25 min 2.43 Injection vol: 4 5 mm: 4 µl 4 15 mm: 12 µl 4 25 mm: 2 µl Sample: Trastuzumab biosimilar (5 mg/ml) Peak label: Resolution

28 Fast ph gradient: 5-min Gradient 15 Fast analysis! Linear ph (compatible with CX-1 ph Gradient Buffers) Column: ProPac Elite WCX, 5 µm Format: 2 5 mm Mobile phase A: CX-1 ph Gradient Buffer A (ph 5.6) Mobile phase B: CX-1 ph Gradient Buffer B (ph 1.2) Gradient: %A %B Flow rate:.8 ml/min Temperature: 3 ºC Detection: UV (28 nm)

29 ph Gradient Method vs icief Method 1 min ph gradient platform method 1 min icief platform method 29

30 icief vs WCX icief ProPac Elite WCX icief ProPac Elite WCX 3

31 Panitumumab (IgG2) Using Salt Gradient 15 Column: ProPac Elite WCX, 5 µm Format: 4 25 mm Mobile phase A: 2 mm MES, ph 5.6 Mobile phase B: 2 mm MES,.5 M NaCl, ph 5.6 Gradient: 12 24% B in 25 minutes Flow rate:.6 ml/min Temperature: 3 ºC Detection: UV (28 nm) Inj. volume: 5 µl (5 mg/ml)

32 Separation of Antibody Drug Conjugate (ADC) Standard 24 Unmodified mab Column: ProPac Elite WCX, 5 µm Format: 4 15 mm Mobile phase A: CX-1 ph Gradient Buffer A (ph 5.6) Mobile phase B: CX-1 ph Gradient Buffer B (ph 1.2) Gradient: 4 6% B in 15 minutes Flow rate: 1. ml/min Temperature: 3 ºC Detection: UV (28 nm) Inj. volume: 5 µl (2.5 mg/ml) 3 ADC

33 Complementary selectivity between ProPac Elite WCX and MAbPac SCX-1 Pertuzumab Trastuzumab 14 ProPac Elite WCX 8 ProPac Elite WCX 12 MAbPac SCX-1 5 µm 8 MAbPac SCX-1 5 µm

34 ProPac Elite WCX vs ProPac WCX-1 Trastuzumab biosimilar Adalimumab biosimilar 7 ProPac Elite WCX, 5µm 1 ProPac Elite WCX, 5µm Similar selectivity Sharper peaks Higher resolution 5 ProPac WCX-1, 1µm 8 ProPac WCX-1, 1µm

35 ProPac Elite WCX 4 15 mm vs ProPac WCX mm Rituximab biosimilar 5 ProPac Elite WCX 4 15 mm 3.42 Faster analysis with similar resolution ProPac WCX mm

36 Part Numbers for Ordering Column Part Number ProPac Elite WCX, 5 µm, 4 25 mm 3325 ProPac Elite WCX, 5 µm, 4 15 mm ProPac Elite WCX, 5 µm, 4 5 mm ProPac Elite WCX, 5 µm, 2 25 mm 3326 ProPac Elite WCX, 5 µm, 2 15 mm 3327 ProPac Elite WCX, 5 µm, 2 5 mm 3328 ProPac Elite WCX, 5 µm, Analytical, 4 25 mm, 3 Columns from 1 Lot 3361 ProPac Elite WCX, 5 µm, Analytical, 4 25 mm, 3 Columns from 3 Lots 3362 ProPac Elite WCX, 5 µm, Analytical, 4 15 mm, 3 Columns from 1 Lot ProPac Elite WCX, 5 µm, Analytical, 4 15 mm, 3 Columns from 3 Lots CX-1 ph Gradient Buffers Part Number 125 ml CX-1 ph Gradient Buffer A ml CX-1 ph Gradient Buffer A ml CX-1 ph Gradient Buffer A ml CX-1 ph Gradient Buffer B ml CX-1 ph Gradient Buffer B ml CX-1 ph Gradient Buffer B 3278 CX-1 ph Gradient Buffer Kit: 125 ml Buffer A ml Buffer B CX-1 ph Gradient Buffer Kit: 5 ml Buffer A + 5 ml Buffer B CX-1 ph Gradient Buffer Kit: 25 ml Buffer A + 25 ml Buffer B