HIGH-THROUGHPUT, SIMPLIFIED PROTEIN PRECIPITATION: A NOVEL 96-WELL FORMAT

FOR PRECIPITATION AND FILTRATION

Kimvan Tran1, Diane M. Diehl1,Brian P. Murphy1

Tim Schultz2, Wilf Wixwat2, Jeff Kane2, Emily Berlin2, Kevin Seeley2 Waters Corporation1, Milford, MA; Pall Life Sciences2, Ann Arbor, MI

OVERVIEW- Protein precipitation is a simple sample preparation method, but can be tedious and difficult to automate. There are several commercially available protein precipitation filter plates, but they suffer from poor performance. Depending on the procedure and the plate, plasma often leaks into the filter before a vacuum is applied, leading to a cloudy filtrate. Other plates suffer from a high percentage of blocked wells. To address these performance issues, we developed a new 96-well protein precipitation and filtration plate that offers a dripless technology with a gradient of purpose-selected filtration media, together with a vented closure to allow for rapid ‘in-well’ sample preparation. We ran a series of ion suppression and extraction studies to ensure the cleanliness of the plate materials. We validated the plate performance for a variety of polar and non-polar acids, bases and neutrals spiked into porcine and rat plasmas. We compared the results to traditional centrifugation methods and other commercially available products. In our studies, we did not observe liquid flow until vacuum was applied. Additionally, less than 1% of the wells plugged — and in most plates we did not see any wells plug. The time for filtration ranges from 30 seconds to 3 minutes, depending on the type and amount of plasma, and the time to process an entire plate of 96 samples is approximately 15 minutes. There is no observable cross talk between the wells. The eluents are free of observable particulates and offer a cleaner eluent than the traditional centrifugation method. This plate out-performs current commercially available products. INTRODUCTION- Pharmaceutical chemists require fast and efficient bioanalytical procedures. Sample preparation is often the rate limiting step and much research and product development has been devoted to increasing the speed of sample preparation. In recent years, sample preparation in the 96-well format has risen in popularity. However, protein precipitation has not been as successfully developed in this format. Many of the commercially available protein precipitation filter plates suffer from poor performance. Depending on the procedure and the plate, plasma often leaks into the filter before a vacuum is applied, leading to a cloudy filtrate. Other plates suffer from a high percentage of blocked wells. To address these performance issues, we developed a new 96-well protein precipitation and filtration plate that offers a dripless technology, a gradient of purpose-selected filtration media, together with a vented closure to allow a rapid ‘in-well’ sample preparation. PLATE DESIGN- The 96-well plate was designed with 1 mL reservoirs. Each well contains a stack of chemically resistant and low extractible membrane filters. The selection of filtration media and the design of the flow path was customized for the protein precipitation purification solvents so that the wells do not empty until vacuum is applied. This allows the precipitation reaction to occur in the filtration plate without the need of a transfer step.

Figure 1.Specially designed and patent-pending vented closures prevent solvent flow until vacuum is applied.

Figure 2. Prototype protein precipitation plate showing the vented closures.

Figure 3. Schematic of the capmat design. Vents in the capmat allow for vortexing and filtration without removal of the capmat — eliminating crosstalk.

MATERIAL SELECTION- An integral part of the design process was the selection of materials for the body of the plate, the vented closures, the capmat and the filtration media. Our requirements were to have ultra-low ion suppression and/or enhancement when using a mass spectrometer (MS) for analysis, as well as ultra-low amounts of extractables as detected by UV or MS. These requirements are crucial for scientists using protein precipitation in the pharmaceutical industry. We developed a series of experiments to test these potential materials. To examine the ion suppression/enhancement effects of the materials, we either soaked the material in acetonitrile or acetonitrile/phosphoric acid (typical PPT crash solvents), or passed the solutions through prototype plates. We set up an HPLC system with a mobile phase containing a series of basic or acidic analytes. The mobile phase for ES+ was 50:50 methanol:water with basic analytes and for ES- was 50:50 methanol:water with acidic analytes. The flow rate was 0.2 mL/min into the electrospray source of a single quadrupole mass spectrometer. With this flow on, we infused 50 µL of the solutions directly into the mass spectrometer by an infusion pump. Blank solutions (i.e. soak solutions without material) were infused first to establish a baseline value for the ion count for each analytes. Then the soak solution itself was infused. Basic Analytes (ES+) m/z Concentration (ng/mL) Propranolol 260 1 Trimethoprim 291 1 Pipenzolate 354 1 Risperidone 411 0.5 Terfenadine 473 1 Methoxyverapamil 485 1 Reserpine 609 5 Ketoconazole 531 1 Acidic Analytes (ES-) m/z Concentration (ng/mL) Fumaric acid 114 1 Malic acid 132 5 Etidronic acid 204 8 Clodronic acid 242 10 Niflumic acid 281 6 Canrenoic acid 357 6 Cholic acid 407 10 Raffinose 503 40 For the extractables studies, two experiments were performed. In the first, the soak solutions were infused into the MS without the HPLC flow on. We ran a full scan to determine what ions were present in the solution. In the second experiment, we made an injection of the soak solution onto an HPLC column and used a photodiode array detector.

Ion Supression - ES+ - Prototype PPT Plate

-50.0%

-40.0%

-30.0%

-20.0%

-10.0%

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

260 291 354 411 473 485 531 609

M ol e c ul e

ACN

Acid/ ACN

I on S upr e ssi on - ES - - P r ot ot y pe P P T P l a t e

-50.0%

-40.0%

-30.0%-20.0%

-10.0%

0.0%

10.0%

20.0%

30.0%40.0%

50.0%

114 132 204 242 281 357 407 503

M ol ecul e

ACN

Table 1.The above plots show the low degree of ion suppression and enhancement in both the ES+ and ES– modes of ionization. This indicates the cleanliness of the PPT plates.

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ACN Blank - 254 nm

ACN Blank - 210 nm

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Prototype – 210 nm

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Prototype – 254 nm

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Competitor Plate C 254 nm

Competitor Plate C 210 nm AU

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A

B

A

B

C

C

Figure A is an injection of ACN onto an HPLC column. Detection is UV at 210 and 254 nm. There are two small peaks in the 210 nm trace. Figure B is an injection of ACN eluent from the Prototype PPT plate. These chromatograms are nearly identical to the ACN blanks. Figure C is an injection of ACN eluent from a commercially available PPT plate. Notice the significant amount of contamination from this plate at both wavelengths.

100 200 300 400 500 600 700 800 900 1000m/z0

100

%

0

100

%

Scan ES+ 1.00e7172.9123

22340608

174.92367422208

245.05364084480

246.99972278656

260.10351579776368.5564

434208

Scan ES+ 1.00e7

ACN Blank

Prototype PlateACN

100 200 300 400 500 600 700 800 900 1000m/z0

100

%

0

100

%

Scan ES+ 1.00e7172.9123

22340608

174.92367422208

245.05364084480

246.99972278656

260.10351579776368.5564

434208

Scan ES+ 1.00e7

100 200 300 400 500 600 700 800 900 1000m/z0

100

%

100 200 300 400 500 600 700 800 900 1000m/z0

100

%

0

100

%

Scan ES+ 1.00e7

0

100

%

Scan ES+ 1.00e7172.9123

22340608

174.92367422208

245.05364084480

246.99972278656

260.10351579776368.5564

434208

Scan ES+ 1.00e7

172.912322340608

174.92367422208

245.05364084480

246.99972278656

260.10351579776368.5564

434208

Scan ES+ 1.00e7

ACN Blank

Prototype PlateACN

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100

%

0

100

%

Scan ES+ 1.00e7152.0208

10146816

156.95185620992

203.98874474368

245.05362617344

263.99571384896 368.6862

750016 842.0283384448

415.5776337088

Scan ES+ 1.00e7

ACN Blank

Competitive Plate CACN

100 200 300 400 500 600 700 800 900 1000m/z0

100

%

0

100

%

Scan ES+ 1.00e7152.0208

10146816

156.95185620992

203.98874474368

245.05362617344

263.99571384896 368.6862

750016 842.0283384448

415.5776337088

Scan ES+ 1.00e7

100 200 300 400 500 600 700 800 900 1000m/z0

100

%

100 200 300 400 500 600 700 800 900 1000m/z0

100

%

0

100

%

Scan ES+ 1.00e7

0

100

%

Scan ES+ 1.00e7152.0208

10146816

156.95185620992

203.98874474368

245.05362617344

263.99571384896 368.6862

750016 842.0283384448

415.5776337088

Scan ES+ 1.00e7

152.020810146816

156.95185620992

203.98874474368

245.05362617344

263.99571384896 368.6862

750016 842.0283384448

415.5776337088

Scan ES+ 1.00e7

ACN Blank

Competitive Plate CACN

Figure 4. The above spectra are the results for the direct infusion of ACN eluents from the Prototype PPT plate and a commercially available PPT plate. In each figure, the top spectra is for the infusion of an ACN blank and the bottom is for the plate eluent. There are a few species from the Prototype plate that are present in low amounts. Clearly, the data on the right for the Competitive plate indicate high levels of contaminants in that plate.

Waters and Micromass, are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. ©2003 Waters Corporation

CONCLUSIONS

• State-of-the-art design ends unpredictable leaking • Maximizes mass spectrometry sensitivity and uptime by eliminating cloudy filtrates • All materials used in manufacturing the plate are highly clean, eliminating ion suppression and

enhancement • Plate design allows for minimized sample cross-talk and maximum sample recoveries

Prototype PPT Plate Flow Results

0.000.501.001.502.002.503.003.50

0 100 200 300

Plasma (µL)

Tim

e (m

in)

Rat PlasmaPorcine Plasma

FLOW RESULTS

Table 2. Flow results indicate that the total time for flow through the plate is related to amount and type of plasma. Porcine plasma filters rather easily in less than 1 minute. Rat plasma flows in under 3 minutes, still quite an acceptable time frame.