Particulates and residues are key types of contamination present in the pharmaceutical industry. These may be produced from an extensive range of sources including undissolved residuals in buffer and media solutions, packaging and various system components such as seals and gaskets. Contamination can also result from side reactions linked to the manufacture of the product, including detergent residues or charred products from maintenance or degradation of the processing equipment such as lubricant oils, metal corrosion or scoring of Teflon gaskets. Particulates are found to contain a variety of materials including glass, aluminum, plastics, rubber and wood-based products.
Particulates can spread to the adjoining air volume of a clean room through airborne contamination or by transport attached to containers or people. This can then lead to contamination of both the manufacturing area and products. These particles, if carried over to the final drug product, can have negative effects such as blockages of blood vessels, impairment of microcirculation, damage to various organs, phlebitis etc. The FDA needs documentation and investigation of unexpected particles or adulterated drugs1, and has taken action against companies that fail to carry out satisfactory investigations for violations involving particle contamination2.
Identifying and understanding the source of particulates is vital for controlling their spread. Once the source is known, then elimination of the particulate contamination becomes significantly simpler. In this regard, SEM-EDX and FTIR-microscopy are powerful tools for identifying particles, and also providing information about size, shape and surface topography. The following analyzes were performed using a FTIR-Microscope (Thermo Nicolet iN10 MX FTIR microscope) and a SEM-EDX (Tescan Vega S 3 LMU with EDAX Octane Plus EDX detector) in order to investigate the chemistry of a brown colored particle found in a steel bioreactor in addition to a brown residue on the surface of the same steel reactor.
SEM-EDX and FTIR microscopy were able to identify the brown residue as iron oxide/iron hydroxide (rust). It also succeeded in determining that the brown particle was made up of a mixture of poly(tetrafluoroethylene) (PTFE, Teflon) and rust (iron oxide/hydroxide). By comparing the results of the brown particle and the brown residue, SEM-EDX and FTIR microscopy were able to prove that particle source was consistent with degradation of the metal container. These techniques were also able to demonstrate that the particle was primarily made up of Teflon, which was correlated with the material specifications to a bigger stirring apparatus made from Teflon.
Objective
The purpose of this analysis was to recognize a brown particle which was present in a bioreactor. A brown residue was also noted in the reactor and it was desired to determine if the particle was chemically related to the residue.
Summary of Results
FTIR-Micro and SEM-EDX results for brown residue and brown particle have been summarized in Table 1. It was found that brown residue (shown in Figure 1) was steady with iron oxide/hydroxide (rust). Brown particle (shown in Figure 1) was found to contain an FTIR spectrum most consistent with a chemical composition of poly(tetrafluoroethylene) (PTFE) and iron oxide. SEM-EDX and FTIR maps acquired of the brown particle are consistent with particles of iron oxide embedded in a PTFE fragment. The chemical composition of the brown particle was found to relate with the material specifications of a bigger stirring apparatus developed from PTFE.
Table 1. Particle Identifications.
| Sample Name |
FTIR Best Match |
SEM Major Elements |
| Brown Residue |
Iron oxide/hydroxide |
Fe, O, Ni |
| Brown Particle |
Iron oxide,/hydroxide PTFE |
F, Fe, C, O |
Figure 1. Optical Micrograph of Brown Particle and Brown Residue.
Figure 2. FTIR micrograph of mapped area.
Figure 3. Overlay of iron oxide absorbances (green) and PTFE absorbances (red).
Figure 4. EDX map overlay of Fluorine (yellow, correlates with PTFE) and Iron (reddish-orange, correlates with iron oxide).
Individual Test Results
A summary of the individual test results has been provided below. All accompanying data, including spectra, has been included in the data section of this report.
Brown Residue
FTIR-Microscopy
Brown residue was received as a brown stain on a metal surface as presented in Figure 1. A portion was removed and then placed on a sampling cell as seen in Figure 5.
Figure 5. Optical Micrograph of Brown Residue on a sampling cell.
Results
It was found that brown residue was consistent with iron oxide/hydroxide. Table 2 provides Specific absorbance assignments for brown residue. An overlay of brown residue and iron oxide/hydroxide can be observed in Figure 6. This identification is further confirmed by the SEM-EDX.
Table 2. FTIR Peaks and Identifications of Brown Residue.
| IR Frequency (cm-1) |
Possible Functional Group |
Possible Source |
| 3480 |
O-H stretch |
Iron Oxide/Hydroxide |
| 1541, 1427, 1345 |
Fe-O comb/overtone |
Iron Oxide/Hydroxide |
| 804 |
Fe-OH stretch |
Iron Oxide/Hydroxide |
| ~675 |
Fe-O stretch |
Iron Oxide/Hydroxide |
Figure 6. Overlay of Brown Residue and an iron oxide/hydroxide standard.
SEM-EDX
Brown residue was examined by SEM-EDX at a control location and one sampling location. Figures 7-8 show the SEM-EDX images and the elemental composition of each sampling location is summarized in Tables 3-4. Figure 9 displays the secondary electron (SE) and backscattered electron (BSE) images of the sample.
Brown residue showed the existence of Fe, Ni and O. This is considered to be consistent with the FTIR identification of iron oxide/hydroxide. The existence of Ni suggests that the reactor composition is consistent with steel.
Table 3. Elemental concentration at area 1 (control) on Brown Residue.
| Element |
Atomic Number |
Series |
Weight % |
Mole % |
% Error |
| Carbon |
6 |
K |
79.06 |
83.41 |
6.28 |
| Oxygen |
8 |
K |
20.94 |
16.59 |
23.72 |
Table 4. Elemental concentration at area 6 on Brown Residue.
| Element |
Atomic Number |
Series |
Weight % |
Mole % |
% Error |
| Carbon |
6 |
K |
17.62 |
33.42 |
15.62 |
| Oxygen |
8 |
K |
32.11 |
45.72 |
15.43 |
| Nickel |
28 |
L |
9.29 |
3.60 |
19.13 |
| Sodium |
11 |
K |
0.27 |
0.27 |
42.12 |
| Aluminum |
13 |
K |
0.26 |
0.22 |
25.74 |
| Chlorine |
17 |
K |
1.13 |
0.73 |
12.12 |
| Iron |
26 |
K |
39.31 |
16.04 |
4.26 |
Figure 7. SEM-EDX image of all sampling locations analyzed of Brown Residue.
Figure 8. SEM-EDX elemental abundance of area 6 analyzed of Brown Residue.
Figure 9. SEM SE (left) and BSE (right) images of Brown Residue.
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Brown Particle
FTIR-Microscopy
Brown particle was received as a white and brown particle on a metal surface as seen in Figure 1. The particle was placed on a sample cell as seen in Figure 10.
Figure 10. Optical Micrograph of Brown Particle on a sample cell.
Results
It was found that brown particle was consistent with a mixture of PTFE and iron oxide. Particular absorbance assignments for brown particle are presented in Table 2. An overlay of the FTIR spectrum of the brown particle in the brown area, the brown residue, and the brown particle in the white area can be seen in Figure 11. An overlay of the FTIR spectrum of the brown particle in the Brown area, PTFE, and the brown particle in the white area can be seen in Figure 12.
It was also observed that the brown areas indicated stronger absorbances consistent with the spectrum from brown residue, while the white areas were more consistent with PTFE. An area map was attained in order to demonstrate the distribution of iron oxide and PTFE in the brown particle. Figures 13-14 display the resulting iron oxide and PTFE heat maps, which specify brown particle is consistent with iron oxide embedded in a PTFE matrix.
Table 5. FTIR Peaks and Identifications Of Brown Particle.
| IR Frequency (cm-1) |
Possible Functional Group |
Possible Source |
| Brown Residue (shown in Table 2) |
Brown Particle Brown area |
Brown Particle White area |
| 3480 |
3371 |
N/O |
O-H stretch |
Brown Residue |
| 1541, 1427, 1345 |
1557, 1426 |
1550, 1448 |
Fe-O comb/overtone |
Brown Residue |
| N/O |
1275-1150 |
1275-1150 |
C-F stretch |
PTFE |
| 804 |
781 |
776 |
Fe-OH stretch |
Brown Residue |
| ~675 |
~675 |
720 |
Fe-O stretch |
Brown Residue |
Figure 11. Overlay of Brown Particle on white area (blue), Brown Particle on brown area (purple), and Brown Residue (red).
Figure 12. Overlay of Brown Particle on white area (blue), Brown Particle on brown area (purple), and PTFE (red).
Figure 13. FTIR heat map of iron oxide absorbances (red is a strong absorbance, blue is weak).
Figure 14. FTIR heat map of PTFE absorbances (red is a strong absorbance, blue is weak).
SEM-EDX
Brown Particle was examined by SEM-EDX at a control location and two sampling locations. A spectrum was taken over a huge area in order to determine the general chemistry of the sample, and a smaller area was attained on a brown portion. Figure 15 displays the secondary electron (SE) and backscattered electron (BSE) images of the sample. It was also observed that one of the bright areas has moved off the particle after the acquisition of an area map as seen in Figure 16. This highlights that one of the bright particles (observed by FTIR to be iron oxide/hydroxide) was not embedded, but instead rested on the surface, and was forced off by electrostatic repulsion when exposed to an intense electron beam. Furthermore, an EDX map was obtained. Individual elemental maps can be seen in Figures 19-Figure 22. Figures 23-26 show the SEM-EDX images and the elemental composition of each sampling location is summarized in Tables 6-8.
It was observed that higher concentrations of F are localized where Fe is not observed as well as the inverse as seen in Figure 17. When this relationship is taken into account with the acquired FTIR map, it can be seen the surface of the PTFE particle is embedded with iron oxide particles since FTIR examines the chemistry of the whole thickness of the sample, while SEM-EDX is considered to be a surface sensitive technique. This is steady with the results observed by FTIR microscopy. An overlay of oxygen and the iron distribution can also be seen in Figure 18, and usually are observed in the same areas of the image signifying that iron is present as iron oxide/hydroxide.
Table 6. Elemental concentration at area 1 (control) on Brown Particle.
| Element |
Atomic Number |
Series |
Weight % |
Mole % |
% Error |
| Carbon |
6 |
K |
74.66 |
79.69 |
7.60 |
| Oxygen |
8 |
K |
25.34 |
20.31 |
22.40 |
Table 7. Elemental concentration at area 3 on Brown Particle.
| Element |
Atomic Number |
Series |
Weight % |
Mole % |
% Error |
| Carbon |
6 |
K |
8.63 |
14.95 |
16.04 |
| Oxygen |
8 |
K |
41.67 |
54.23 |
15.39 |
| Fluorine |
9 |
K |
15.76 |
17.27 |
15.65 |
| Sodium |
11 |
K |
1.40 |
1.27 |
7.68 |
| Aluminum |
13 |
K |
0.14 |
0.11 |
34.83 |
| Silicon |
14 |
K |
0.16 |
0.12 |
30.85 |
| Sulfur |
16 |
K |
0.16 |
0.10 |
34.22 |
| Chlorine |
17 |
K |
0.38 |
0.22 |
19.00 |
| Iron |
26 |
K |
26.30 |
9.81 |
5.56 |
| Nickel |
28 |
K |
5.40 |
1.92 |
11.52 |
Table 8. Elemental concentration at area 3 on Brown Particle.
| Element |
Atomic Number |
Series |
Weight % |
Mole % |
% Error |
| Carbon |
6 |
K |
24.06 |
35.02 |
23.86 |
| Oxygen |
8 |
K |
18.59 |
20.32 |
25.75 |
| Fluorine |
9 |
K |
44.06 |
40.55 |
16.94 |
| Nickel |
28 |
L |
11.46 |
3.41 |
13.05 |
| Aluminum |
13 |
K |
0.38 |
0.24 |
14.05 |
| Iron |
26 |
K |
1.45 |
0.45 |
23.69 |
Figure 15. SEM SE (left) and BSE (right) images of Brown Particle before mapping.
Figure 16. SEM SE (left) and BSE (right) images of Brown Particle after mapping.
Figure 17. EDX map overlay of Fluorine (yellow) and Iron (reddish-orange).
Figure 18. EDX map overlay of Oxygen (green) and Iron (reddish-orange).
Figure 19. EDX map of Carbon.
Figure 20. EDX map of Fluorine.
Figure 21. EDX map of Oxygen.
Figure 22. EDX map of Iron.
Figure 23. SEM-EDX image of all sampling locations analyzed of Brown Particle.
Figure 24. SEM-EDX elemental abundance of area 1 (control) analyzed of Brown Particle.
Figure 25. SEM-EDX elemental abundance of area 2 analyzed of Brown Particle.
Figure 26. SEM-EDX elemental abundance of area 3 analyzed of Brown Particle.
Analysis Conditions
This section of a Jordi report presents information on the methods employed including instrument type, solvents, sample preparation, temperatures, etc. The particular conditions have been removed for this case study.
Closing Comments
Deformulation of an unknown material is proposed to provide a best estimate of the chemical nature of the sample. All chemical structures are supported by the evidence that is present but are subject to revision upon receipt of further evidence. Supplementary factors such as material processing conditions could also affect final material properties.
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References
1 FDA Regulation 21 CFR 211 Subpart E
2 FDA fines drug manufacturer $500 million for violations including insufficient investigation of rejected lots http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/EnforcementActivitiesbyFDA/ucm118411.htm
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