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List Of SOPs

1.0 Purpose:

Batch culture of the CHO DP12 cell line for the production of recombinant human Anti IL-8 monoclonal antibodies. Cells will be cultured in 100ml spinner flask and scaled up to 1L in a bioreactor.

2.0 Scope: 

Applies to the production of recombinant Anti IL-8 monoclonal antibodies from recombinant Chinese Hamster Ovary (CHO) DP12 clone.

3.Responsibilities:

  1. It is the responsibility of the course instructor/lab assistant to ensure that this SOP is performed as described and to update the procedure when necessary.

  2. It is the responsibility of the students/technician to follow the SOP as described and to inform the instructor about any deviations or problems that may occur while performing the procedure.

4.References:

  1. CHO DP12-ATCC® CRL-12444/12445 cell line construction culture method https://www.atcc.org/products/all/CRL-12445.aspx

5.Precautions:

  1. Use BL2 safety measures and discard waste in biohazard containers.

  2. Routine care should be exercised in the handling of buffers and samples of biological materials, which may have harmful biological activity in the case of accidental ingestion, needle stick etc.

  3. Gloves, a lab coat and protective eyewear should be worn when handling buffers and samples.

6.Equipment and Materials:

  1. Equipment

    1. Biological safety cabinet

    2. CO2 incubator

    3. Fisher Scientific Isotemp Low speed magnetic stirrer

      1. Clean and autoclaved 100 ml Bellco spinner flasks

  1. Thermo Scientific Biomate UV-visible recording spectrophotometer

  2. Thermo Scientific Evolution 220 UV-vis spectrophotometer

  3. Oakton PC 700 Bench Series pH/Conductivity/°C/°F Meter

  4. Fisher Scientific Isotemp 37°C water bath

  5. Fisherbrand microcentrifuge

  6. Nikon E100-LED Compound Light Microscope with 100X magnification (10X objective lens)

  7. Hemocytometer with cover glass

  8. Luna-FL Fluorescence cell counter and Acridine Orange/Propidium iodide stain

  9. Biorad iMark Microplate reader

  10. Applikon EZ-control bioreactor controller with A 3-liter glass autoclave bioreactor and the processor

  11. 500 ml and 1L liquid addition/feed bottles

  12. 250 ml glass feed bottle for 150 ml alkaline solution

  13. 100 ml glass bottle

  14. Sterile sample bottle

  15. Male and female autoclave connectors

  16. Tubing clamps

  17. Gas filters, 0.2µm

  18. Autoclavable silicone tubing size 14(1.6mm interior diameter)

  19. Autoclavable silicone tubing size 16(3.1mm interior diameter)

  20. Autoclavable silicone tubing size 25(4.8mm interior diameter)

  21. Laboratory gasses: Air compressor, CO2, O2(optional)

  22. Materials:

    1. Vials of CHO DP12 cells (ATCC CRL-12445/12444)

    2. Dublecco’s Modified Eagle’s Medium (DMEM) Corning # 10-013 CV

    3. Superlow IgG Fetal Bovine Serum (Hyclone # SH3089802)

    4. Insulin-Transferrin Selenium (ITS-G) 100X (Gibco # 41400-045)

    5. 0.2mM methotrexate stock solution (1000X) in PBS

    6. Nalagene 250 ml 0.2 µm filter units

    7. Trypan Blue (0.4% solution)

    8. 1X PBS

    9. 150 ml of 1M NaHCO3 (sodium bicarbonate)

    10. 10mg/ml gentamycin

    11. O2 Electrolyte solution for DO probe

    12. Control serum for glucose and lactate assay

    13. Glucose oxidase assay kit

    14. Lactate assay kit

    15. Glucose standard

    16. Lactate standard

    17. 100ml and 250 ml graduated cylinder

    18. Sterile serological pipettes (2ml, 5ml, 25 ml, and 50 ml)

    19. Pipette aid

    20. Spectrophotometer UV/Vis cuvettes and cuvette rack

    21. Oakton pH 4.0 and pH 7.0 standard buffers

    22. 50 ml beakers

    23. 1-T25 vented tissue culture flask for blank

    24. Test tube rack

    25. 1.5 ml microfuge tube and tube holder

    26. P20, P200, and P1000 micropipettes and compatible tips

    27. Sterile 250ml glass bottles for storage of CHO cell media

    28. Sterile sample bottles and associated size 16 about 12 cm length

    29. Aluminum foil

    30. Autoclave tape

    31. Cotton

7.Procedure:

The batch record should be completed step by step by operator of the task and the verifier of the task.

  1.    Preparation of CHO DP-12 Complete Growth media- DMEM, 90% Super low IgG Fetal Bovine Serum, 10% 1X Insulin-Transferrin Selenium (ITS-G), 200nM methotrexate solution.

    1. Prepare biological safety cabinet per Labconco Purifier Class 2 Biological Safety cabinet (BSC) Operation SOP

    2. Gather the following items, spray with 70% isopropanol, and place in the biological safety cabinet.

      1. Pipette aid (wipe with paper dampened with 70% IPA)

      2. 5ml sterile pipettes

      3. 25ml sterile pipettes

      4. 500ml bottle of pre-sterilized DMEM media

      5. Super low IgG Fetal Bovine Serum

      6. 250 ml sterile 0.22 µm filter unit

    3. 120 ml Complete Growth media:

      1. Add 108 ml of DMEM, 12 ml of Super Low IgG FBS, 1.2 ml ITS-G (100X), and

0.120 ml methotrexate (1000X) to the top portion of the filter unit and sterile filter

  1. Preparation of Spinner flask and Blank tube


1.Purpose:

To produce a batch culture of bacterial host cells

2.Scope:

Applies to the production of green fluorescent protein from recombinant E. coli cells

3.Responsibilities:

  1. It is the responsibility of the course instructor/lab assistant to ensure that this SOP is performed as described and to update the procedure when necessary.

  2. It is the responsibility of the students/technicians to follow the SOP as described and to inform the instructor about any deviations or problems that may occur while performing the procedure.

4.References:

  1. LB Broth manufacturer instructions

  2. LB Agar manufacturer instructions

  3. autoclave SOP

  4. shaking incubator SOP

  5. water bath SOP

  6. spectrophotometer SOP

  7. incubator SOP

  8. pH meter SOP

  9. Gram stain SOP

  10. microscope SOP

5.Definitions: N/A

6.Precautions:

Recombinant E. coli is a BL2 microorganism.Use BL2 safety measures and discardwastein biohazard containers.

7.Materials:

  1. 1mL vials of E.coli recombinant for GFP (-86°C freezer)

  2. Luria-Bertani (LB) Broth premixed powder (room temp)

  3. Luria-Bertani (LB) Agar premixed powder (room temp)

  4. Arabinose (room temp)

  5. Ampicillin powder (4-8°C)

  6. 70% Isopropanol (room temp)

  7. deionized water

  8. small beaker

  9. 30cc syringe

  10. sterile syringe filter (0.2µm)

  11. sterile 50mL centrifuge tube

  12. 500 mL Erlenmeyer shake flask with cap

  13. 2L Erlenmeyer flask with cap

  14. petri dishes (100x15mm, approx. 55 per batch of LB agar)

  1. 125 mL glass bottle with cap

  2. 25mL, 100mL, 250mL, 1000mL graduated cylinders

  3. magnetic stir plate

  4. magentic stir bars

  5. autoclave tape

  6. laboratory film such as Parafilm

  7. sterile pipettes (25mL, 2mL) and pipet pumps

  8. 1.5mL microfuge tubes

  9. cell Spreader

  10. spectrophotometer Cuvettes

  11. balance

  12. autoclave

  13. 55°C water bath

  14. 37°C radial shaking incubator

  15. spectrophotometer

  16. micropipettors and sterile pipette tips

  17. micro-scale pH meter (pH7 and pH4 commercially prepared buffers)

  18. microscope with 1000x magnification

  19. Gram stain reagents

8.Procedure:

  1. Solution and Media Preparation

    1. Culture Broth: Luria-Bertani (LB) Broth, Ampicillin (0.09mg/mL), Arabinose (1.8mg/mL)

      1. Gather the following items and place on a clean lab bench area: 500mL Erlenmeyer shake flask with cap

125mL glass bottle with cap 250mL graduated cylinder

magnetic stir bar and magnetic stir plate autoclave tape

  1. Weigh out approximately:

LB Broth premix             4g

arabinose                         0.36g

  1. Add LB broth premix and arabinose to a clean 500mL shake flask.

  2. Measure about 200mL of deionized water using a 250mL graduated cylinder and add to the LB/ARA broth.

  3. Stir to dissolve the ingredients using a magnetic stir bar and stir plate.

  4. Measure approximately 100mL of the LB/ARA broth using the 250mL graduated cylinder and transfer to the 125mL glass bottle.

  5. Remove the stir bar.

  6. Place caps on 500mL shake flask and 125mL bottle loosely to allow air flow (but not so cap can fall off). Place a small piece of autoclave tape on each.

  7. Label shake flask: LB/ARA, [date], [initials], GFP, [group #].

  8. Label glass bottle: LB/ARA, [date], [initials], GFP, [group #].

​For Complete SOP with Annexures - Click on Read more, Subscribe and Download         


1.0 Purpose

Operation of the BioLogic LP Chromatography System

2.0 Scope

Applies to the BioLogic LP Chromatography System for purifying proteins

3.Responsibilities:

  1. It is the responsibility of the course instructor/lab assistant to ensure that this SOP is performed as described and to update the procedure when necessary.

  2. It is the responsibility of the students/technicians to follow the SOP as described and to inform the instructor about any deviations or problems that may occur while performing the procedure.

4.References:

  1. BioLogic LP Chromatography System Instruction Manual

5.Definitions:

  1. CV: Column Volume; CV = (L in cm)[(radius of column in cm) 2]

  2. L = Length of column (meaning the height of the bead bed)

  3. HETP: Height Equivalent to Theoretical Plate; HETP = L/N

  4. N = 5.54 (tR/w1/2)2

  5. tR: retention time

  6. w1/2: peak width at half height

  7. h: Reduced Plate Height; h = HETP/Dp

  8. Dp: bead diameter

6.Precautions: N/A

7.Materials:

  1. deionized Water

  2. Equilibration Buffer A (Refer to the process SOP)

  3. Equilibration Buffer B (Refer to the process SOP)

  4. Cleaning Solution (Refer to the process SOP)

  5. biopure water

  6. container for waste fluid

  7. collection tubes for fraction collector or collection containers

  8. column (Amicon Vantage-L Biochromatography column and accessories)

  9. resin (Refer to the process SOP.)

  10. lab towels

8.Procedure:

  1. Turn on BioLogic LP system (switch is in the front, on the lower left side of the system).

  2. Turn on computer.

  3. Click on the LP DataView icon.

  4. Verify that the computer is communicating with the system as indicated by a green “Receive” circle on the upper right side of the computer screen.

8.5.Pump Calibration

  1. Based on the desired flow rate, select the appropriate tubing for the pump as follows:

Flow rates of 0.04-0.8 mL/min require 0.8mm tubing. Flow rates of 0.2-4.0 mL/min require 1.6mm tubing. Flow rates of 0.8-15.0 mL/min require 3.2mm tubing.

  1. Verify that the correct tubing is in the pump.

    1. Remove the platen by lifting the grey handle (Figure 2).

    2. If necessary, insert the correct tubing.

    3. Replace platen and lock into place.

    4. If tubing was replaced readjust the platen and recalibrate the pump.

      1. Loosen the platen adjust screw located on the top of the pump (Figure 2) by turning counterclockwise until there is slight resistance.

      2. Tighten the platen screw clockwise the appropriate number of COMPLETE turns.

0.8mm tubing requires 5 turns

1.6mm tubing requires 4 turns

3.2mm tubing requires 3 turns

  1. Recalibrate the pump.

    1. Press the MANUAL mode key.

    2. Press the PUMP instrument key.

    3. Select FLOW, then select CALIBRATE.

    4. Select the appropriate tubing size.

    5. Select NOMINAL.

8.6.Purge System with Buffer A and Zero the UV Monitor

  1. Place each buffer line into a container filled with Buffer A (Equilibration Buffer).

  2. Attach the column inlet tube directly to the column outlet tube.

  3. Press the MANUAL mode key.

  4. Select BUFFER.

  5. Select MIX.

  6. Type in 50% B.

  7. Select OK.

  8. Select PURGE.

  9. Allow system to purge until conductivity reading on the display panel of the Biologic LP system controller stabilizes (less than 5 minutes).

  10. Select BUFFER.

  11. Using the arrow key, select C.

  12. Select OK.

  13. Allow system to purge until conductivity reading on the display panel of the controller stabilizes (less than 5 minutes).

​For Complete SOP with Annexures - Click on Read more, Subscribe and Download         


1.Purpose:

To describe the proper gowning procedure for personnel entering the biomanufacturing suite to minimize the number of particles and viable microorganisms in the suite

2.Scope:

Applies to gowning performed in the gowning area prior to entering the biomanufacturingsuite.

3.Responsibilities:

  1. It is the responsibility of the Supervisor to ensure that this SOP is performed as described and to update the procedure when necessary.

  2. It is the responsibility of the all employees to follow the SOP as described and to inform the instructor about any deviations or problems that may occur while performing the procedure.

4.References:

Gowning for Aseptic Filling

5.Definitions: N/A

6.Precautions:

70% isopropyl alcohol is flammable and poisonous if ingested. Avoid creating excessive mist when using spray bottles with IPA.

7.Materials:

  1. disinfecting hand soap

  2. sterile 70% (v/v) isopropyl alcohol (IPA)

  3. head cover

  4. hood (if needed)

  5. facial hair cover (if needed)

  6. cleanroom coverall, sterile Tyvek

  7. shoe covers

  8. non-powdered nitrile gloves

  9. sterile gloves

  10. lab tissues such as Kimwipes

  11. boots

  12. hood

8.Procedure:

  1. Employees should wear clean clothes that are not overly capable of shedding particulates (i.e., wool sweaters).

  2. Gowning must occur only when no one is entering or exiting the gowning area. Likewise, do not enter the gowning area while someone is gowning.

  3. Wearing makeup and jewelry is prohibited in the cleanrooms. If necessary, remove makeup and jewelry before proceeding to the Pre-Gowning Area.

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