The Latest RBD 3000 Applications Advanced Analytical User

1
The Latest RBD 3000 ApplicationsAdvanced
Analytical Users MeetingNovember 30,
2006Chicago, IL
2
Overview

• Contamination Level Test
• For materials that contain low-level bio-burden
• Determines that material meets microbial
  specification
• Utilizes the Most Probable Number (MPN) protocol
• Useful for complex matrices
• Antimicrobial Effectiveness Test
• Rapidly screens candidate preservative systems
• Tracks an increase in dead cell count

3
Contamination Level Test Feasible in a Variety
of Complex Matrices

• Company A In-process pharmaceutical product
• Company B Various nutraceutical finished
  products
• Juice, Body Butter, Fiber
• Company C Liquid nutraceutical finished product
• Company D Various alcoholic beverages

4
CLT Protocol for lt100cfu/g specification
5
CLT Results Company A
In-process Product Specification lt1000cfu/g
Spiked Below Specification
Sample Dilution RBD 3000 Result ( pos./ neg.) Plate Result ( pos./ neg.) Microbial level determined by RBD 3000 (cfu/g)
110 3/0 3/0 gt1
1100 3/0 3/0 gt10
11,000 3/0 3/0 gt100
110,000 1/2 1/2 lt1,000
Product contains gt100cfu/g and lt1000cfu/g
product is within specification Actual spike was
559 cfu Ps. aeruginosa/g product
Spiked Above Specification
Sample Dilution RBD 3000 Result ( pos./ neg.) Plate Result ( pos./ neg.) Microbial level determined by RBD 3000 (cfu/g)
110 3/0 3/0 gt1
1100 3/0 3/0 gt10
11,000 3/0 3/0 gt100
110,000 3/0 3/0 gt1,000
Sample Dilution Theoretical Result Detection by RBD 3000 Growth on YM PetrifilmTM
1100
11,000
110,000 - - -
Product contains gt1000cfu/g product fails
Actual spike was 3920 cfu Ps. aeruginosa/g
product
6
CLT Results Company B
Body Butter Juice Products Specification
lt100cfu/g or mL
Body Butter Spiked Below Specification
Sample Dilution RBD 3000 Result ( pos./ neg.) Growth on YM PetrifilmTM ( pos./ neg.) Microbial level determined by RBD 3000 (cfu/g)
1100 2/1 2/1 gt10
11,000 0/3 0/3 lt100
110,000 0/3 0/3 lt1000
Product contains gt10cfu/g and lt100cfu/g product
is within specification Actual spike was 35 cfu
C. albicans/g body butter
Juice Spiked Above Specification
Sample Dilution RBD 3000 Result ( pos./ neg.) Growth on YM PetrifilmTM ( pos./ neg.) Microbial level determined by RBD 3000 (cfu/g)
1100 3/0 3/0 gt10
11,000 2/1 2/1 gt100
110,000 1/2 1/2 lt1000
Sample Dilution Theoretical Result Detection by RBD 3000 Growth on YM PetrifilmTM
1100
11,000
110,000 - - -
Product contains gt100cfu/g product fails Actual
spike was 110cfu C. albicans/mL juice
Taken from poster presentation at ASM 2006 A
Rapid Most Probable Number Test for Yeast in
Nutraceutical Products, K.A. Molitor and A.M.
Steger, Advanced Analytical Technologies, Inc.,
Ames, IA 50010 and D. Wright and M. Roblin,
Morinda, Inc., Orem, UT 84604
7
Contamination Level Test Benefits

• 18-24hr time-to-result
• Detects lower levels of potential microbial
  contamination than the traditional plate count
  method in complex/opaque matrices
• Results are confirmed by the Most Probable Total
  Count by Multiple-Tube Method in USP Chapter
  lt61gt, Microbial Limits Test

8
Antimicrobial Effectiveness Test
9
Antimicrobial Effectiveness Test

• Used to screen candidate preservatives for liquid
  products
• Pharmaceutical and Cosmetic Industries routinely
  perform
• New reformulated products
• Problems with the current method
• Compendial method takes 35 days
• Requires significant labor materials
• Not all types /or formulations of antimicrobials
  are screened due to time labor constraints
• Delays development of suitable preservative
  systems
• Delays product time to market

10
Antimicrobial Effectiveness Test Solution

• Use the RBD 3000 to rapidly screen candidate
  preservative systems
• Benefits
• Shorter time to result
• Significant labor and materials savings
• Evaluate more types/formulations of
  antimicrobials
• Develop better preservative systems
• Speed product time to market

11
Antimicrobial Effectiveness Test Protocol

• Challenge 0.01 (w/v) Benzoic Acid with 105 106
  cfu/mL
• Perform challenge studies with Ps. aeruginosa, S.
  aureus, and C. albicans
• Use RBD 3000 to determine titer of inoculum
• Sample at 0, 2hr, 4hr, 6hr, 8hr 24hr
  post-inoculation
• Dilute samples 110 in phosphate buffer for RBD
  3000 Biomass Dead Cell counts using the Biomass
  and Dead Cell Test Kits
• Dilute samples in phosphate buffer 4 Tween 20
  for TSA plate count comparison

12
0.01 Benzoic Acid Challenged with Ps. aeruginosa
Test 1
13
0.01 Benzoic Acid Challenged with Ps. aeruginosa
Test 2
14
0.01 Benzoic Acid Challenged with S. aureus
15
0.01 Benzoic Acid Challenged with C. albicans
16
Antimicrobial Effectiveness Test Action Items

• Test other preservative systems
• Determine utility in various liquid product
  formulations
• Demonstrate feasibility with A. niger spores

17
Summary

• Contamination Level Test
• Provides a rapid sensitive detection method for
  determining if materials meet particular
  microbial specifications
• Antimicrobial Effectiveness Test
• Provides a rapid method for screening candidate
  preservative systems