Pharma Tips

Lyophilizer - Standard Operating Procedure

By: Pharma Tips | Views: 3543 | Date: 02-Oct-2011

The process of using a laboratory scale lyophilizer presents a number of unique hazards. These hazards include but are not limited to extreme pressure changes, a potential for glassware to explode or implode, and the possibility of frostbite or tissue damage associated with exposure to cryogenic materials. The following information contains written Standard Operating Procedures for the safe use of a laboratory lyophilizer.

The process of using a laboratory scale lyophilizer presents a number of unique hazards. These hazards include but are not limited to extreme pressure changes, a potential for glassware to explode or implode, and the possibility of frostbite or tissue damage associated with exposure to cryogenic materials. The following information contains written Standard Operating Procedures for the safe use of a laboratory lyophilizer.

Lyophilizer

BACKGROUND
Biological materials typically must be dried to stabilize them for storage, preservation, or shipping. Many times, drying causes damage and some loss of cellular or protein activity. Lyophilization, also called freeze-drying, is a method of sample preservation that significantly reduces damage to biological samples.

Substances that are not damaged by freezing can usually be lyophilized so that refrigerated storage is unnecessary. Important exceptions are mammalian cells, nearly all of which are destroyed by lyophilization. Many microorganisms and proteins survive lyophilization well, and it is a preferred method of drying vaccines, pharmaceuticals, blood fractions, and other laboratory samples. Some specialist food products are also lyophilized. They rehydrate easily and quickly because of the porous structure left after the ice sublimes.

AUTHORIZED CONTACT PEOPLE
Below is a list of people ‘authorized’ to train or assist others on the use of the lyophilizer. ‘Authorized’ means at least one quarter of regular, independent use of the lyophilizer (including the liquid nitrogen trap) within the past year. Undergraduate researchers or individuals from other laboratories are responsible for reading and understanding this SOP, but are not responsible for training others in safety procedures, regardless of experience.

Equipment users will seek and accept training or assistance only from one of these contact people.

Authorized Lyophilizer Contact People:
1) 
2) 
3) 
4) 
5) 

SAFETY ESSENTIALS

1. WEAR APPROPRIATE EYE PROTECTION AT ALL TIMES WHEN WORKING WITH OR ANYWHERE NEAR A LYOPHILIZER.
2. FOR MEDICAL EMERGENCIES, FIRES, OR EXPLOSIONS, CALL 911 IMMEDIATELY AND REPORT THE DETAILS OF THE INCIDENT.

EQUIPMENT DESCRIPTION

See attached copy of manual for complete description and basic operating instructions.
 

There are two major components to the operating procedure:
1) Lyophilizer Use
2) Liquid Nitrogen Trap Use
All users must be familiar with both of these components, even if they never intend to use the liquid nitrogen trap themselves. All users must sign the form attached to this procedure to indicate that they have reviewed and understand these procedures.


1) Lyophilizer Use
a) Inspection of glassware & seals
b) Freezing of sample in liquid nitrogen (includes hazards of liquid nitrogen)
c) Placing of sample on lyophilizer and evacuating vessel
d) Repressurizing/removing sample (includes flask failure)
e) Maintenance (oil changing and refrigerator coil cleaning)

a) Inspection of glassware and seals
Use only glassware designed for the lyophilizer!!
The lyophilizer, when in good working order, should have a pressure of 5-50 millitorr (normal atmospheric pressure is 760 torr), as indicated on the LED screen. (If the pressure is outside of this range, contact one of the people listed above.) This means that all glassware attached and under vacuum on the lyophilizer represents a significant implosion hazard.

All glassware used on the lyophilizer must be free of any visible defect (cracks, chips, or scratches), no matter how seemingly minor. Any glassware that is defective in this way must not be used under any circumstances. If such glassware is found, it should be discarded. The seals themselves are somewhat more forgiving, in that a defective seal reduces the vacuum. However, defective seals should still be removed from service.

A greater risk is present in seals that connect two pieces of glass. All seals of this type place rubber between joined pieces of glass. If these seals are used improperly, the glass pieces come into contact and scratch each other during installation. These scratches compromise the integrity of the glass thus creating a potentially serious implosion hazard. Install any seals of this type carefully to avoid this; if you are uncertain of how to do this do not proceed until you have received further training from one of the contact people given above.

b) Freezing the sample in liquid nitrogen
Liquid nitrogen is a hazardous material. Liquid nitrogen boils at 77 Kelvin (-194 Celsius). As such, it presents a severe frostbite hazard. When using liquid nitrogen, wear proper eye protection and protective gloves. Proper eye protection will depend on the splash hazard, but should at a minimum include chemical safety glasses. If larger volumes of liquid nitrogen are being use or if the potential for a splash is greater, chemical goggles and a face shield may be necessary. NEVER WEAR A FACE SHEILD WITHOUT PROPER GLASSES OR GOGGLES. Protective gloves should be approved for liquid nitrogen splash hazards and are typically designated CRYO gloves by manufacturers. Be aware, however, that the gloves offer only very transient protection, in that if liquid nitrogen soaks into the material they will freeze themselves, becoming the hazardous agent! Remove any gloves or articles of clothing that become saturated with liquid nitrogen. In addition, as it boils and attains the gas phase, liquid nitrogen displaces all oxygen in the vicinity. Therefore, in closed spaces liquid nitrogen is an asphyxiation hazard. Never use liquid nitrogen in an area that is not well ventilated.
Liquid nitrogen will condense oxygen. Oxygen boils at 79 Kelvin (-192 Celsius). Therefore, oxygen will condense out of the air and into any environment cooled by liquid nitrogen. Do not use styrofoam containers to hold liquid nitrogen because they are likely to become brittle (see below) and also present an extreme fire/explosion hazard. Condensing liquid oxygen is a severe explosion hazard when using the liquid nitrogen trap, as discussed in that section.
Finally, because of the very low temperature, liquid nitrogen renders most materials extremely brittle, and places them under extraordinary thermal stress. Place liquid nitrogen only into those containers designed for the purpose of holding cryogenic material. See the contact people above for guidance on which containers qualify for this use if you are unsure.
Freezing the sample. Place the sample container into the liquid nitrogen. If it is necessary to hold the sample in the nitrogen by hand, it is best to use forceps (big tweezers). When using the silvered-glass evacuated dewars, be aware that the dewars themselves present an implosion hazard. Do not place more samples into the dewar than can fit easily. The nitrogen will splash around quite a bit; this is normal. Wear gloves and eye protection! Remove the samples periodically and check to make sure the samples are completely frozen before proceeding to the next step. Don’t put the sample higher than your head when you do this (as in, trying to hold it up to the light to see inside). If the sample is very cold, you can ‘carry’ a little bit of condensed air along and inadvertently drop it onto your head or (presumably protected) eyes. Alternatively, you can wait until the bubbling in the nitrogen comes to nearly a complete stop (this means that the sample is at roughly the same temperature as the nitrogen. It is generally necessary to replenish the nitrogen several times during the freezing process. Always refill the smaller dewars from an appropriate container, e.g. the 4L liquid nitrogen dewar, and not from the 160L/240L cylinder. When the sample is frozen, transfer the sample to an appropriate lyophilizer flask just prior to placing it on the lyophilizer.


c) Placing the sample on the lyophilizer and evacuating the sample
This is fairly straightforward. Using the appropriate seals for the flask that is being used, attach the sample to the lyophilizer and evacuate the flask. When doing this, make sure the seal is fitted properly to the flask before evacuation. The sample will not thaw if taken directly from the nitrogen to the lyophilizer in the time it takes to check the seal carefully, so there is no reason to rush this. It is recommended that you evacuate the flask slowly at first, to reduce (slightly) the risk of implosion. However, a flask in proper working condition will not require this, and the implosion, if it occurs, is likely to be swift and violent. Therefore, wear eye protection while installing the sample, and any time you are in ‘line of sight’ of the lyophilizer!


d) Repressurizing/removing the sample
Inspecting the vessel for any cracks that may have developed as you approach, place one hand on the vessel or seal (assuming no cracks are evident), and with the other hand gently restore the valve to the closed position. Do not allow the glassware to bump into any surface on the lyophilizer during this time. Wear eye protection! Once the valve is in the closed position and any ‘whooshing’ sound from the in-rushing air has stopped, the flask may be removed from the lyophilizer.
In the event that cracks have developed in any glassware attached to the lyophilizer, turn all switches on the front of the machine to the off position. The point here is that one of them is the switch for the vacuum pump, and you should not take any time in reading which is which---just turn them all off. Then, leave the lab and evacuate anyone else in the lab with you. Notify people in adjacent labs of the hazard, by leaving the room and using the hallway or other safe alternate route to get to the other lab(s). If, in your best judgement, you do not believe that you are able to safely turn off the lyophilizer, then simply leave the area at once, and evacuate any other people in the lab. It is far better to have the pump running after an implosion with no one around than to suffer severe injury trying to prevent the implosion. NOTE:  By signing this SOP, you indicate that you are willing and capable of following at least one of these courses of action. If this is not the case, do not sign this SOP and do not use the lyophilizer.


e) Maintenance
See attached document for lyophilizer pump maintenance procedures. Only trained individuals are to perform maintenance procedures. If you believe that the lyophilizer or any components require maintenance, see one of the contact people with your concerns.

2) Liquid Nitrogen Trap Use
a) Who needs to use the liquid nitrogen trap?
b) Who needs to learn about the liquid nitrogen trap?
c) Initial trap condition
d) Preparing the trap
e) Placing of trap on lyophilizer , and inside dewar
f) Evacuating trap (prior to adding liquid nitrogen)
g) Adding liquid nitrogen to the dewar
h) Attaching sample flask to the trap
i) Removing sample flask from trap
j) Removing trap from lyophilizer
k) What to do if oxygen is condensed in the trap

a) Who should use the liquid nitrogen trap?
Anyone who is attempting to remove any solvent from a sample except pure water must use the liquid nitrogen trap. This includes solvents with higher melting points than water (e.g. DMSO), since these solvents fall outside the usual day-to-day operations. In this way, the cleanup is kept much simpler in that those who service the machine know what is and isn’t on the coil when we clean it. Non-volatile buffers may be used without the trap; volatile buffers (e.g. ammonia, acetate) must be lyophilized with the trap. If you are unsure if you need the trap, ask one of the contact people.

b) Who needs to learn about the liquid nitrogen trap?
All users must learn about the liquid nitrogen trap, even if they will never use it. The reason is that the trap presents a significant hazard to all users, and may be used frequently.

c) Initial trap condition
The trap should be clean and dry before you use it. If it is not clean and dry, then first try to find the person that last used it, so that they can clean it out or at least tell you what is in it. Otherwise, find one of the contact people to assist you.

d) Preparing the trap
The liquid nitrogen trap is a custom-made piece of glassware, but its construction is essentially the same as a standard cold-finger. The most significant addition is a teflon greaseless stopcock, which controls the vacuum between the sample and the trap. Make sure that this stopcock is closed before proceeding. The main joint and ball fittings (male and female) are ground glass, and need to be greased. USE ONLY SILICONE BASED GREASE FOR THIS PURPOSE. USING APIEZON OR OTHER HYDROCARBON-BASED GREASE CAN RESULT IN A HIGH RISK OF EXPLOSION!!! The explosion risk comes from condensing liquid oxygen, which can happen accidentally. Liquid oxygen can form explosive compounds when exposed to oil, grease, or hydrocarbons. See section 1-b above and 2-k below. The main joint is fitted with a Keck clamp after the two pieces of the trap are connected at the main joint.

e) Placing trap on lyophilizer, and inside dewar
The installation of the trap on the lyophilizer is somewhat involved; all people using the trap must be shown the proper techniques for installing and removing the trap by one of the contact people above. As always, eye protection is required. The trap is held on a ring-stand using a large standard clamp holding the trap by the outside of the main ground-glass joint. After attaching the clamp to the trap, the clamp, trap and clamp-holder (which holds the clamp to the ring-stand) are removed as a single unit from the ring-stand. A 4-liter liquid nitrogen dewar is placed on the ring-stand, and the trap re-attached to the ring-stand such that the trap is now inside of the dewar. The trap should be as low as possible in the dewar without touching the bottom. A glass male-male ball joint connector is used to hook up the trap to the lyophilizer, and is held in both places with Keck clamps. Make sure again that the greaseless teflon stopcock is in the closed position before evacuating the trap.

f) Evacuating the trap (prior to adding liquid nitrogen)
Evacuate the trap as you would a normal lyophilization flask. Take care to use two hands, and not to disturb the connection between the lyophilizer and the trap.  The trap must be evacuated before liquid nitrogen is added to the dewar.  FAILURE TO DO SO WILL RESULT IN THE CONDENSATION OF OXYGEN IN THE TRAP, PRESENTING A SEVERE EXPLOSION HAZARD!!!  Wait until the pressure reading is below 100 millitorr before adding liquid nitrogen to the dewar. 

g) Adding liquid nitrogen to the dewar
Follow the same recommendations as for freezing samples (section 1-b) in liquid nitrogen while adding the nitrogen. Again, eye protection is required.

h) Attaching sample to the trap
Follow the same guidelines as for adding a sample to the lyophilizer, with one additional caution. Because of the way the trap is designed, the flask becomes a closed system for a short time when the joint of the sample apparatus is in contact with the trap, and before the vacuum is opened to the sample. This can sometimes result in liquid nitrogen and/or condensed air that has traveled with the frozen samples now pressurizing the flask, which makes it difficult to form a good seal before applying the vacuum. Avoid this by inverting the frozen samples (to pour out condensed gasses) and allow them to stand for about 30-60 seconds before adding the sample to the trap. Also, one can apply the sample with one hand and open the stopcock with the other at the moment of contact. Secure the sample apparatus to the trap with a Keck clamp, and prop the sample from below 


i) Removing the sample from the trap
Close the greaseless stopcock, and remove the sample apparatus slowly, allowing the flask to repressurize gradually. Follow the same warnings as for removing ordinary samples (section1-d). At this point, the trap is still under vacuum, and the pressure reading should remain steady.

j) Removing the trap from the lyophilizer
Perform this step smoothly without interruption, as there is no way to avoid having the trap immersed in liquid nitrogen for a short time without vacuum and open to the air. As has been stressed, this presents a risk of condensing oxygen. The process of removing the trap should take no longer than 60 seconds . During this short of a time no significant amount of oxygen will condense. Open the valve on the lyophilizer that leads to the trap, as for a normal sample. Disconnect the connector arm from both the trap and lyophilizer, and set aside. Use one hand to hold the trap and clamp, and with the other disconnect the clamp-holder from the ringstand, so that the clamp-holder, clamp and trap can be lifted smoothly in one vertical motion from both the stand and the dewar. Do not allow the trap to bump into the bottom of the dewar at this time. Move the trap, clamp and clamp-holder to a second nearby ring stand and attach.


k) What to do if oxygen is condensed in the trap
If liquid oxygen is discovered by a person not on the contact list above when the trap is removed from the liquid nitrogen, then the user should follow the procedure given below until one of the contact people can be notified. At that time, the user should evacuate themselves and anyone else from the lab, and keep the lab empty until help arrives.
At 77 Kelvin, just about the only thing that will remain liquid besides nitrogen is oxygen. Assume if you see liquid in the trap, and you see that the trap is obviously cold (e.g. just taken from liquid nitrogen), that liquid oxygen is present. Liquid oxygen has a characteristic blue or blue-gray hue, whereas nitrogen is colorless. Some solvents, such as ethers and methylene chloride, have very low boiling points and therefore persist as liquids for a very long time in liquid nitrogen traps. However, it is always safer to assume the worst when dealing with the trap. If liquid oxygen is believed to be present, the flask should be mounted on the nearest ring stand located away from any potential ignition source and left alone until the trap comes to room temperature. If possible, mount the trap in the hood; but in any event do not spend any time looking for the ‘perfect’ place to put it---the priority is to put the trap down safely and evacuate the area!
Once the trap has arrived at room temperature, the explosion hazard is still present!!! Empty any liquid into a clean beaker (for disposal later), and flush the still-assembled trap 5 times with large amounts of water. This will remove the oxygen-rich atmosphere and the explosion hazard with it.

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