TITLE: Antibody lyophilization
AUTHORS: Christine Camacho, Marc Bosse, Sean Bendall, Mike Angelo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the lyophilization process of stock antibodies using an overnight lyophilization process.</div></div>

[STEPS]
?. [Preparation]
If processing a large amount of vials, pre-cool extra aluminum tube racks for at least  at .
-80 °C
?. [Preparation of 5 µg Antibody Aliquots ]
Select antibodies to lyophilize using data provided on MIBItracker.
?. [Preparation of 5 µg Antibody Aliquots ]
Measure the total volume of each antibody to lyophilize using 100-1000µl pipette and record volume. Alternatively, use the total volume (including the antibody stabilizer) recorded in the MIBItracker.
?. [Preparation of 5 µg Antibody Aliquots ]
Add records on the calculation Excel sheet: Conjugation ID, Target name, Channel, Concentration, Total volume of labeled antibody to lyophilizeCalculation sheet will give you: a) number of vials of 5 µg b) volume of 500 mM trehalose to add to get 100 mM final c) volume of ultrapure water complete volume of master mixIDTargetChannelConcentration (µg/mL)Total volume (µL)#vialsTrehaloseH2O1615NeuN154200791=E2*D2/5000=F2*20=(F2*100)-(E2+G2)
IDTargetChannelConcentration (µg/mL)Total volume (µL)#vialsTrehaloseH2O1615NeuN154200791=E2*D2/5000=F2*20=(F2*100)-(E2+G2)
?. [Preparation of 5 µg Antibody Aliquots ]
Using the calculation sheet, prepare and label tubes prior to start.  Prepare an ice bucket with dry ice and cooled aluminum rack.
?. [Snap Freeze Samples]
Transfer the samples to for at least 1 hour. Samples can be held at this step at  for longer if queued for vacuum chamber. Vials may be capped and stored in plastic cryoboxes if desired.
-80 °C
-80 °C
?. [Lyophilization]
When ready to dry samples, ensure frozen vials are placed into aluminum rack. Place loaded rack with vials into chamber.
?. [Lyophilization]
On the lyophilizer screen menu, press the vacuum tab and start the vacuum. You can record pressure and temperature parameters by pressing the folder icon tab and name a new logging file starting with the date format YY/MM/DD followed by the initial of the user (ex. 180117MB).
?. [Finish process and Storage]
Store at
4 °C
?. [Preparation]
Thaw on ice the necessary amount of frozen aliquot 500 mM trehalose solution, or prepare solution fresh
?. [Preparation]
On the lyophilizer screen menu, press the collector tab and start the collector.
?. [Finish process and Storage]
Allow process to run for 24 hours. End process by selecting "Collector" -> STOP and "VACUUM" -> STOP from the menu. Before collecting samples from vacuum chamber cabinet, obtain new caps for tubes.
?. [Finish process and Storage]
Open vacuum release valve and open the vacuum chamber. Remove samples. Immediately cap vials with new caps to prevent moisture from getting into the sample.
?. [Preparation]
To prepare fresh trehalose 500 mM solution, in a 50mL conical falcon tube, add  of trehalose and  of Ultrapure water.
1.89 g
10 mL
?. [Preparation]
Filter with a syringe filter unit (0.1 µm membrane). Store remaining solution in 1 mL aliquots at .
-20 °C
?. [Preparation of 5 µg Antibody Aliquots ]
Aliquot mixture into tubes using automatic pipette. Cap tubes and place into cooled rack to snap freeze. Once you have snap frozen the samples they must be kept frozen.
?. [Lyophilization]
Ensure release vacuum valve is closed (valve is located on back left side of chamber cabinet). Maximum of two shelves in chamber are allowed. Maximum of 4 x 90 vial racks allowed.
?. [Label templates]
Templates for the tube labels and dots for the caps can be found here:
?. [Lyophilization]
NOTE: If message appears for "Collector Moisture", simply plug hose/bottle located on the left of the machine into the  plug located at the left front. Moisture will automatically drain into waste collection bottle attached to the hose. Unplug collection setup prior to running collector.