Fast IEF Method Development Using Whole-Column Detection cief

Transcription

1 Fast IEF Method Development Using Whole-Column Detection cief Jiaqi Wu 27 Coronet Road, Toronto, Ontario, CANADA M8Z 2L8

2 Conventional cief (cief Performed on Commercial CE Instruments) - + ph gradient Focusing OH - H + ph9 ph6 ph4 Mobilization Carrier ampholytes Protein zones - Detection point + Capillary column OH - H + H + ph6 ph4

3 Conventional cief Advantages Uses existing commercial CE instruments Provides quantitative protein analysis Disadvantages Long analysis time (about 1 run/hour) Long method development time (weeks) Non-reproducible peak pattern and low resolution due to mobilization process

4 cief without Mobilization Whole-Column Detection Detector: CCD Imaging Camera in UV + - Voltage Voltage Outlet H + OH - IEF Column Focused Zones Inlet Capillary Dialysis Hollow Fiber Light Beam at 280 nm

5 Whole-Column Detection cief ice280 Analyzer Definition CE analyzer specially designed for cief ice280 Analyzer Advantages Provides quantitative protein analysis Short analysis time (up to 6 runs/hour) Accelerated method development (days) Reproducible peak patter and high resolution Wide pi range (ph 2-11)

6 cief Method Development Where Should I Start? Carrier ampholytes (CAs) Manufacturer ph range Use of mixture of narrow and wide ph range carrier ampholytes Concentration Sample concentration Additives Focusing voltage and time

7 Carrier Ampholytes Brand Names Pharmalytes (Pharmacia) Ampholines (Pharmacia) Servalyts (Serva) Biolytes (Bio-Rad) Others may be one of the above, but are sold under different commercial names.

8 Carrier Ampholytes Concentration 2 8% reported ph range wide range for unknown samples UV absorption at 280 nm Pharmalytes have uniform absorption along the whole ph range Start from 4% ph3-10 Pharmalytes for unknowns Different carrier ampholytes may give slightly different resolutions for different samples. Try different ones if the resolution is not satisfactory.

9 Sample Concentration Start from 0.1 mg/ml in final solution for samples with one major peak Start from (number of major peak X 0.1) mg/ml for samples with multiple major peaks * Major peaks are peaks that have 20% of total peak area

10 Additives Methyl cellulose (for all samples) Modify hydrophobic surface of FC coated column Enhance resolution by reducing sample s diffusion coefficient Additives for stabilizing proteins during IEF (optional) Increase sample solubility and stability (sugars, non-ionic or zwitterionic surfactants, and urea of <4M) Denature protein samples (>8 M urea) Additives for keeping proteins reduced during IEF (optional) DTT

11 Initial Conditions Carrier ampholytes 4% ph3-10 Pharmalytes Sample concentration 0.1 mg/ml for single major peak sample, >0.1 mg for samples with multiple major peaks Additives 0.35% methyl cellulose Focusing voltage and time ~500 v/cm 6 min for wide ph range carrier ampholytes TIP: If you have a gel IEF method for your sample, start from there.

12 Narrow ph Range Carrier Ampholytes Use narrow ph range carrier ampholytes to improve resolution when necessary The narrow ph range carrier ampholytes can be used alone or mixed with wide ph range carrier ampholytes When narrow ph range carrier ampholytes are used, use the same electrolytes as those used for wide ph range carrier ampholytes When you use carrier ampholyte mixtures, try ratio: 1:1 5:1 (narrow ph range CAs:wide ph range CAs)

13 pi Calibration and Peak Identification Using pi Markers Why are pi markers required? ph gradient can be squeezed by salts in samples Different salt concentrations in samples due to different dilution factors Samples from different sources may have different salt concentrations in their matrices Using pi markers is a reliable way to compensate for the salt effect

14 pi Markers for cief Protein pi markers Available from major reagent companies Multiple peaks May interact with samples pi may change in different matrices Small molecule pi markers (preferred for cief) Single peak High sensitivity at 280 nm (~1 µg/ml gives enough signal) pi does not change in most matrices Available from Convergent Bioscience and other vendors

15 Method Development Flow Diagram Adjust sample concentration Initial condition: 0.1mg/ml 4% ph3-10 Pharmalytes, 6 min Yes If Abs<0.05 or > Adjust focusing time 2. Try additives No No Peak pattern reproducible? Yes Select two pi markers and determine sample pi 1. Narrow range ampholytes or mixture of carrier ampholytes Yes Need higher resolution? No Results

16 Method Development Example 1 -Mab Sample Run #1 Initial Conditions: 0.1 mg/ml sample concentration 4% ph 3 10 Pharmalyte 6 min focusing Results: Sample concentration is enough Sample pi is around 8 To be Tested: Peak pattern reproducibility Sample pi Absorbance ph Peak Position (mm) ph10

17 Method Development Example 1 -Mab Sample Runs #2 and #3 New Conditions: The initial conditions pi markers 7.5 and 9.6 Results: Peak pattern reproducible Sample pi = 8.4 To be Tested: Possible resolution enhancement Absorbance pi marker Peak Position (mm) pi marker 9.6

18 Method Development Example 1 -Mab Sample Run #4 New Conditions: 3% ph Pharmalyte 1% ph3-10 Pharmalyte Results: Resolution is enhanced Satisfactory result A b so rb an ce Peak Position (mm)

19 Method Development Example 1 -Mab Total sample runs: 4 Total time: About 1 hour

20 Method Development Example 2 - Glycosylated Protein Sample Run #1 Initial Conditions: 0.1 mg/ml sample concentration 4% ph 3 10 Pharmalytes 6 min focusing Results: Sample concentration is low Sample pi is around 7 To be Tested: Higher concentration Peak pattern reproducibility Enhancing resolution Sample pi Absorbance ph Peak Position (mm) ph10

21 Method Development Example 2 - Glycosylated Protein Sample Runs #2 and #3 New Conditions: 0.5 mg/ml sample concentration 3% ph 5-8 Pharmalytes 1% ph 3 10 Pharmalytes Two pi markers (5.3, 7.9) Conclusion: Sample concentration is right Peak pattern is reproducible Sample pi is determined Satisfactory resolution pi marker 5.3 pi marker

22 Method Development Example 2 - Glycosylated Protein Total sample runs: 3 Total time: About 45 min

23 Method Development Example 3 -Virus Sample Run #1 Initial Conditions: 0.1 mg/ml sample concentration 4% ph 3 10 Pharmalytes 6 min focusing Results: Sample concentration is too high To be Tested: Lower concentration Peak pattern reproducibility

24 Method Development Example 3 -Virus Sample Runs #2, #3, #4 New Conditions: 0.01 mg/ml sample concentration 4% ph 3 10 Pharmalytes Two pi markers (4.4, 7.7) Results: Sample concentration is right Sample pi = 6.4 Peak pattern nonreproducable due to aggregation To be Tested: Peak pattern reproducibility pi marker 4.4 pi marker 7.7

25 Method Development Example 3 -Virus Sample Runs #5 - #13 New Conditions: 0.01 mg/ml sample concentration 4% ph 3 10 Pharmalytes 1 M, 2 M and 4 M urea (3 conditions) three runs under each condition Results: Peak pattern reproducable in 1 M urea Satisfactory result

26 Method Development Example 3 -Virus Total sample runs: 13 Total time: About 4 hour