Methods for identification of particles in a fluid sample
A method of distinguishing between proteinaceous and non-proteinaceous particulates in a fluid sample includes the steps of acquiring a brightfield background image of a membrane filter, introducing a fluid sample onto the membrane filter, acquiring a brightfield image of filtered particles resting on the membrane filter, generating a particle mask based on the brightfield background image and the brightfield image of filtered particles, introducing a fluorescent dye onto the membrane filter, detecting fluorescence on the particle mask, and distinguishing between proteinaceous and non-proteinaceous particulates based on the detected fluorescence. A method for detecting other types of particles, such as polysorbate particles, silicone oil or protein monomers is also disclosed.
1. A method of distinguishing between proteinaceous and non-proteinaceous particulates in a fluid sample comprising:
acquiring a brightfield background image of a membrane filter while the membrane filter is free of a fluid sample;
introducing a fluid sample onto the membrane filter;
acquiring a brightfield image of filtered particles resting on the membrane filter;
generating a particle mask based on the brightfield background image and the brightfield image of filtered particles;
introducing a fluorescent dye onto the membrane filter;
detecting fluorescence on the particle mask; and
distinguishing between proteinaceous and non-proteinaceous particulates based on the detected fluorescence.
2. The method of claim 1 further comprising:
generating a total particle distribution, a protein particle distribution, and a non-protein particle distribution based on the distinguishing.
3. The method of claim 1 further comprising:
generating an image of protein and non-protein particles, wherein the protein and non-protein particles are different colors.
4. The method of claim 1 further comprising:
ignoring data outside the particle mask for the steps of detecting and distinguishing.
5. The method of claim 1 further comprising:
acquiring a background fluorescence and removing baseline fluorescent intensity.
6. The method of claim 1 , wherein the step of introducing a fluorescent die takes place immediately after the step of introducing a fluid sample onto the membrane filter.
7. The method of claim 1 , wherein the fluorescent dye is introduced to the fluid sample prior to the step of introducing the fluid sample onto the membrane filter.
8. The method of claim 1 , wherein the membrane filter is a track etched membrane.
9. The method of claim 1 , wherein the membrane filter is dyed with a fluorescence photo absorber.
10. The method of claim 9 , wherein the photo absorber comprises sudan black dye.
11. The method of claim 1 , wherein the membrane filter comprises a polycarbonate or polyester.
12. The method of claim 1 , wherein the membrane filter is coated with PVP.
13. The method of claim 1 , wherein the step of introducing a fluorescent dye comprises introducing between 5 and 50 uL of dye.
14. The method of claim 1 , wherein the fluid sample is bounded by a hydrophobic ring disposed on a well plate.
15. The method of claim 1 further comprising:
individually and separately imaging a plurality of filtrate samples bounded by a plurality of hydrophobic rings disposed on the well plate.
16. A method of distinguishing between particulates in a fluid sample comprising:
acquiring a brightfield background image of a membrane filter while the membrane filter is free of a fluid sample;
introducing a fluid sample onto the membrane filter;
acquiring a brightfield image of filtered particles resting on the membrane filter;
generating a particle mask based on the brightfield background image and the brightfield image of filtered particles;
introducing a fluorescent dye onto the membrane filter;
detecting fluorescence on the particle mask; and
distinguishing between particulates based on the detected fluorescence.
17. The method of claim 16 , wherein the fluorescent dye is selected for detecting protein particles.
18. The method of claim 7 , wherein the fluorescent dye is a protein aggregate dye.
19. The method of claim 17 , wherein the fluorescent dye is a monomer dye.
20. The method of claim 16 , wherein the fluorescent dye is selected for detecting polysorbate particles.
21. The method of claim 16 , wherein the fluorescent dye is selected for detecting silicone oil.
22. The method of claim 16 , wherein the fluorescent dye is selected for detecting protein monomers.
23. The method of claim 16 further comprising:
ignoring data outside the particle mask for the steps of detecting and distinguishing.
24. The method of claim 16 further comprising:
acquiring a background fluorescence and removing baseline fluorescent intensity.
25. The method of claim 16 , wherein the membrane filter is dyed with a fluorescence photo absorber.
26. The method of claim 25 , wherein the photo absorber comprises sudan black dye.