High-Throughput, Simplified Protein Precipitation: A Novel 96-Well Format for Precipitation and Filtration EAS 2003

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1 Kimvan Tran 1, Diane M. Diehl 1, Tim Schultz 2, Wilf Wixwat 2, Brian P. Murphy 1, Jeff Kane 2, Emily Berlin 2, Kevin Seeley 2, Ziling Lu 1 1 Waters Corporation, Milford, MA 2 Pall Life Sciences, Ann Arbor, MI Pall Life Sciences High-Throughput, Simplified Protein Precipitation: A Novel 96-Well Format for Precipitation and Filtration EAS 2003

2 Outline Introduction Product Development Sirocco Protein Precipitation Plate Design and Attributes Performance Summary

3 Introduction Protein precipitation (PPT) is a quick and dirty method for sample preparation in the pharmaceutical industry Various methods are utilized Commercial PPT plates exist, but many are difficult to use and suffer from poor performance To facilitate faster and better PPT, Waters has co-developed an new PPT plate with Pall Life Sciences a valued technical partner

4 Market Research Surveyed customers doing protein precipitation They talked about: Labor intensive sample prep (to feed the LC/MS) Equipment intensive steps Looking for relief in sample prep real time savings. Eliminate steps that would save real time, I would pay a reasonable premium. So why aren t more scientists using current PPT plate technologies?

5 Problems with Existing Plates Tend to leak before customer was ready to vortex Precipitate in the filtrate Plates suffer from plugging Cross contamination Some manufacturers recommend crashing the protein in tubes and transfer to the filter plate. This creates extra steps and transfers (i.e. time). Binding on glass fiber filters Extractables/leachables that interfere with analyses, especially in LC/MS n

6 Development Criteria No cross contamination Minimize the levels of: Leaking customer controls flow Extractables/leachables Ion suppression/enhancement Additional Concerns Smaller volumes of plasma need greater recovery of supernatant (without drawing precipitate) Precipitate fouling HPLC column

7 New Sirocco Protein Precipitation Plate 96-well, 1 ml/well filtration plate Contains a valve mat on the tips which open and allow flow under vacuum* Stack of membranes, smallest is 0.45 µm (nylon-based) Vented cap mat Product 5 pack 5 filter plates 5 vented cap mats in separate bag Pall Life Sciences *patent pending

8 Drawing of Plate Partial Valve Mat Shows 8 rows with valve mat 4 rows of tip only

9 Single Valve There is a slit at the bottom of the valve that opens under 3-4 Hg vacuum

10 All Materials Extensively Tested for Ion Suppression/Enhancement and Extractables Ion suppression or enhancement is NOT observed with any of the materials, including the final plate configuration Levels of extractable materials are negligible and orders of magnitude cleaner than the competitive plates Examined both by UV and MS Let s take a look at some data.

11 Ion Suppression/Enhancement Protocol* Two MeOH/water mobile phase solutions were prepared One with 8 basic analytes, m/z , ES+ One with 8 acidic analytes, m/z , ES- HPLC flow rate was 0.2 ml/min of above mobile phases into a single quadrupole mass spectrometer Candidate materials were soaked in ACN and ACN:acid ACN and ACN:acid were flowed through prototype plates With HPLC pump on, these material solutions were infused into the MS source via a syringe pump Monitored the m/z of the mobile phase analytes and compared responses to blank solutions for degree of ion suppression or enhancement *Mallet, C. R.; Lu, Z.; Mazzeo, J. R. Rapid Commun. Mass Spectrom. 2003; in press.

12 ES+ Data for Sirocco Plate Ion Supression - ES+ - Sirocco PPT Plate 50.0% 40.0% Suppression or Enhancement (%) 30.0% 20.0% 10.0% 0.0% -10.0% -20.0% -30.0% -40.0% ACN Acid/ACN -50.0% Molecule

13 ES- Data for Sirocco Plate Ion Supression - ES- - Sirocco PPT Plate 50.0% 40.0% Suppression or Enhancement (%) 30.0% 20.0% 10.0% 0.0% -10.0% -20.0% -30.0% -40.0% ACN -50.0% Molecule

14 UV Data - Sirocco 2003 Waters 0.20 Corporation AU ACN Blank nm 0.00 AU Minutes ACN Blank nm Minutes AU Sirocco 254 nm AU Minutes Sirocco 210 nm Minutes

15 UV Data Competitive Plates AU Plate A 254 nm 0.00 AU Minutes Plate A 210 nm 0.00 AU Minutes Plate B 254 nm 0.00 AU Minutes Plate B 210 nm Minutes

16 Extractables Data 100 % % Scan ES+ 1.00e7 ACN Blank Plate B ACN Scan ES+ 1.00e7 100 % % Scan ES+ 1.00e7 ACN Blank Scan ES+ 1.00e7 Plate A ACN 0 m/z m/z 100 % % Scan ES+ 1.00e7 ACN Blank Scan ES+ 1.00e7 Sirocco ACN m/z

17 Eliminates Cloudy Filtrates Plate A Plate B Precipitate in the tips Precipitate in the filtrate Sirocco Consistently clean filtrates, no precipitate in the tips.

18 Protocol Set the Sirocco plate on a collection plate. This keeps the valve tips suspended in wells of collection plate during processing. 1. Add crash solvent (typically acetonitrile with ISTD) 2. Rapidly inject plasma into wells 3. Apply vented cap mat (use a roller for good seal) 4. Vortex PPT plate/collection plate stack at a medium speed ensures complete mixing and precipitation for 1 minute. 5. Filter on vacuum manifold at 8-10 Hg. Note: Never remove cap mat or valve tips this eliminates cross talk between wells. The entire assembly is disposed after filtration.

19 Plasma Flow Times Sirocco Plate Flow Results Time (min) Rat Plasma Porcine Plasma Plasma (µl)

20 Recovery Data 300 µl ACN with internal standard added to plate (N = 6) Rat plasma (100 µl) spiked with 10 pq/µl of 8 analytes Vortexed and filtered for 3 minutes % Recovery RSD (%) Propranolol Labetalol Atenolol Nadolol Metoprolol Alprenolol Pindolol Oxprenolol

21 Summary Novel design of the Sirocco Protein Precipitation Plate enables faster and more reliable protein precipitation Simple protocol Leak-free No cross contamination Faster processing No ion suppression or enhancement Minimal levels of extractables/leachables Clean filtrates

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