1 | P a g e

Goal: Develop a system that enables 19 or fewer vials to be utilized for protocol development. Theory: A small batch will freeze dry faster than a large batch in a lab freeze dryer. First Experimental Set: A full tray in a lab freeze dryer and then run 19 vials in the same dryer using the same cycle (using CM-Pirani convergence to indicate end of primary drying).

Run a REVO with a full tray of vials and time Experimental Design 3ml fill in a 10 ml vial Water Freezing step ramp 0.5C/min to-40C, hold 60 mins Primary drying shelf set point +20C Vacuum set point 100 mT

Run a REVO with a 19 vials and time Experimental Design 3ml fill in a 10 ml vial Water Freezing step ramp 0.5C/min to -40C, hold 60 mins Primary drying shelf set point +20C Vacuum set point 100 mT

REVO Full Tray 636 minutes in primary

drying

REVO 19 Vials 512 minutes in primary

drying

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REVO 19VAC-PREVO 19VAC-CREVO FULLTRAY VAC-PREVO FullTray VAC-C

Full tray CM and Pirani convergence

Results REVO full tray vs 19 vials

Experimental Group # 1

Water

Full tray in REVO

Vs 19 Vials in

REVO

19 vial CM and Pirani convergence

636 minutes is the target for all other water runs

Results

19 vials dry quicker than

a full tray based on

end of primary

drying being detected by CM vs Pirani convergence

Results: 19 Vials Take less time. Why? Assumption: Radiant energy has a significant effect on the freeze drying process via edge vial effect. Smaller batch runs are more susceptible to this effect because a larger percentage of the vials are “edge effect vials”.

2 | P a g e

Experimental Group # 1

5% Sucrose

Full tray in

REVO

Run a REVO with a full tray of vials and time Experimental Design 2ml fill in a 10 ml vial 5% sucrose by weight Freezing step -10 hold 30 mins, -40C hold 120 mins Primary drying shelf set point -20C Vacuum set point 60 mT

REVO Full Tray 2026 minutes in primary

drying

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REVO2ml fill in 10ml vial

5% sucrose1 tray filled

TP1 TP2 TP3 TP4 TP5 TP6 TP7

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Results

Full tray took 2026

mins based on end of primary

drying being detected by CM vs Pirani convergence

2026 minutes is the target for all other sucrose runs

3 | P a g e

Theory: Eliminate radiant energy, and thus edge effect vials, and your small batch runs will be more like the larger batch runs.

Second Experimental Set: Eliminate radiant energy from the freeze dryer when using 19 vials. Build a small freeze dryer with the ability to control wall temperature. Note primary drying time.

Made controlled temperature walls. Also tried adding commercial insulation and aluminum shield to minimize edge effect.

ALPHA design

Run ALPHA with a 19 vials and wall at +20C Experimental Design 3ml fill in a 10 ml vial Water Freezing step ramp at 0.5C/min to -40C, hold 30 mins Primary drying shelf set point 20C Vacuum set point 100 mT

Run ALPHA with a 19 vials and wall at -20C Experimental Design 3ml fill in a 10 ml vial Water Freezing step ramp at 0.5c/min to -40C, hold 30 mins Primary drying shelf set point 20C Vacuum set point 100 mT

ALPHA with +20C Walls 532 minutes in primary

drying. (reminder: target is 636 mins)

ALPHA with -20C Walls 557 minutes in primary

drying (reminder: target is 636 mins)

Experimental Group #2

Water

Eliminate

radiant energy to minimize edge effect

vials by cooling and/or

shielding the chamber walls of the freeze

dryer.

Results

The minimization

of radiant energy does

not eliminate edge effect.

4 | P a g e

Run ALPHA with a 19 vials and wall at +20C Experimental Design 2ml fill in a 10 ml vial 5% sucrose by weight Freezing step -10 hold 30 mins, -40C hold 120 mins Primary drying shelf set point -20C Vacuum set point 60 mT

ALPHA, wall at +20C 789 minutes in primary

drying

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Alpha UnitWalls at +20C

2 ml in 10 ml vial5% sucrose

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Array and thermocouple placement in

Alpha

Experimental Group #2

5% Sucrose

Eliminate

radiant energy to minimize edge effect

vials by cooling and/or

shielding the chamber walls of the freeze

dryer.

5 | P a g e

ALPHA, wall at -20C 1096 minutes in primary

drying

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Results: Cooling the chamber wall to -20C appeared to make some difference but the elimination of radiant energy does not appear to be the answer to simulating a larger freeze dryer. Why?

Run ALPHA with a 19 vials and wall at -20C Experimental Design 2ml fill in a 10 ml vial 5% sucrose by weight Freezing step -10 hold 30 mins, -40C hold 120 mins Primary drying shelf set point -20C Vacuum set point 60 mT

Experimental Group #2

5% Sucrose Continued

6 | P a g e

Theory: Eliminating radiant energy did not appear to eliminate edge effect in the 19 vial array. Taking a closer look at how the individual vials are responding may help us understand this further. The common perception is that when we eliminate radiant energy the edge effect should be eliminated.

Third Experimental Set: Utilizing the gravemetric measurement method determine the rate of freeze drying by various vials

within the array. Do perimeter vials freeze dry differently than center vials even minimal radiant energy is present?

Run ALPHA with a 19 vials and wall at -40C Experimental Design 3ml fill in a 10 ml vial Water Freezing step at 0.5 to -40C, hold 30 mins Primary drying shelf set point +20C Vacuum set point 100 mT

Run ALPHA with a 19 vials and wall at -40C with aluminum shield and additional insulation. (10+ runs conducted) Experimental Design 3ml fill in a 10 ml vial Water Freezing step at 0.5 to -40C, hold 30 mins Primary drying shelf set point +20C Vacuum set point 100 mT

% dry by weight 25% of the way

into primary drying.

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Experimental Group #3

Water

Run systems with wall at -40C. Study the rate of

sublimation in individual vials

in a freeze dryer that has

eliminated radiant energy to gain a better perspective on

what is happening in

the dryer.

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Results: Edge effect NOT eliminated by lowering the temperature of the chamber wall. If the presumption that radiant energy caused edge effect and radiant energy is eliminated, then edge effect should be eliminated. This experiment does not verify this

assumption. Gravemetric studies indicate that edge vials are drying faster than the other vials, even when there is minimal radiant energy

Results

Low temperature

chamber walls does not eliminate

“edge effect”

Std Dev 4.9 Std Dev 4.4

7 | P a g e

Run ALPHA with a 19 vials and wall at -40C

Experimental Design 2ml fill in a 10 ml vial 5% sucrose by weight Freezing step -10 hold 30 mins, -40C hold 120 mins Primary drying shelf set point -20C Vacuum set point 60 mT

ALPHA, wall at -40C 1042 minutes in primary

drying

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Alpha Wall at -40C2 ml fill in 10 ml vial

5% sucrose

TP1 TP2 TP3 TP4 TP5 TP6 TSHELF_S Vacuum-P Vacuum-c

Experimental Group #3

5% Sucrose

Run systems with wall at -40C. Study the rate of

sublimation in individual vials

in a freeze dryer that has

eliminated radiant energy to gain a better perspective on

what is happening in

the dryer.

Results

Low temperature

chamber walls does not eliminate

“edge effect” as evidence by run time not approaching

REVO run time of 2026 mins

Gravemetric tests not yet conducted.

Warning: At -40C in these conditions the wall starts behaving

like an internal condenser

8 | P a g e

Theory: The product vials have a thermal effect on one another (essentially acting as cold sinks and hot sinks). Simulate adjacent vials by directly contacting the edge vials and controlling the temperature.

Fourth Experimental Set: Find a way to contact the cold temperature walls to the outside edge vials. If “edge effect” is eliminated this indicates that vial to vial contact has a significant effect in “edge effect”.

No longer using the Alpha unit. Designed and build the MicroFD with

“thermal simulator” temperature controlled blocks.

Run MicroFD with a 19 vials and wall at -40C Experimental Design 3ml fill in a 10 ml vial Water Freezing step ramp 0.5 to -40C, hold 30 mins Primary drying shelf set point +20C Vacuum set point 100 mT

MicroFD with simulator blocks (aka LyoSim™) 633 minutes in primary drying

ONLY 3 MINUTES DIFFERENT THAN THE FULL TRAY RUN DONE IN THE REVO

EDGE EFFECT ELIMINATED ALL VIALS NOW BEHAVE LIKE CENTER VIALS

The use of thermal simulator blocks in the MicroFD created an environment where the sublimation rate was very similar (essentially the same) in every vial. The elimination of edge effect indicates the MicroFD

can be utilized for protocol development for larger freeze dryers based on its ability to simulate center vials.

Experimental Group # 4

Water

Adjacent vials

affect the thermal

characteristics of one another. Since edge vials

do not have the same

number of adjacent vials

they sublimate differently.

Have the controlled

temperature wall come in contact with the vials to

eliminate edge effect.

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Std Dev 4.4

9 | P a g e

(see specific experiments for additional details)

Experiment #

Product Conditions (see experiments for details)

Time Vial variation in sublimation rate

1 Water REVO full tray 636 mins Not studied

1 Water REVO 19 vials 512 mins High disparity, data not reported here

2 Water Alpha 19 vials +20C walls 532 min High disparity, data not reported here

2 Water Alpha 19 vials -20C walls 557 mins High disparity, data not reported here

3 Water Alpha 19 with -40C wall Not timed High disparity, edge effect seen (see pg 3)

3 Water

Alpha 19 vials with -40C wall and additional radiant energy

shielding

Not timed High disparity, edge effect seen (see pg 3)

4 Water MicroFD with LyoSim (thermal sink emulators

touching vials)

633 mins (successfully

simulates REVO run with full tray

Variation in vials minimized, edge effect eliminated

(see pg 4)

Summary

Water

Patent # 9121637 Accuflux Heat flux sensor in a freeze dryer LyoSim Patent Pending

10 | P a g e

Additional & Subsequent Studies. Big Pharma Collaboration: As part of our on-going work we conducted a blind study with a large pharma group. They created a cycle in a SP Scientific LyoStar. They supplied us with vials and product that had a critical temperature of -24C. Within 3 runs we were able to fine tune the Micro FD to simulate their LyoStar freeze dryer run to their satisfaction. They have subsequently purchased a unit for further study.

Experiment #

Product Conditions (see experiments for details)

Time Vial variation in sublimation rate

1 5% Sucrose

REVO full tray 2026 mins Not yet studied

2 5% Sucrose

Alpha 19 vials +20C walls 789 mins Not yet studied

2 5% Sucrose

Alpha 19 vials -20C walls 1096 mins Not yet studied

3 5% Sucrose

Alpha 19 with -40C wall 1042 mins Not yet studied

4 5% Sucrose

MicroFD with LyoSim (thermal sink emulators

touching vials)

Not yet studied Not yet studied

Product Experiments

with customer

Summary

5% Sucrose