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. 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 #].

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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).

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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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SOP for Purification of Anti IL-8 Monoclonal Antibody from CHO DP12 Conditioned Media using Gravity Flow Protein A Chromatography

1.0 Objective:

To describe the procedure for the purification of anti-IL-8 monoclonal antibody (mAb) expressed in CHO DP12 cells using gravity flow Protein A affinity chromatography.

2.0 Scope:

This SOP applies to the downstream purification team involved in the capture of IgG monoclonal antibodies from clarified CHO DP12 conditioned media using gravity-based Protein A resin columns.

3.0 Responsibility:

• Downstream Manufacturing Technicians: Perform the purification procedure as outlined.
• Quality Assurance: Review and approve the documentation and ensure compliance.
• Supervisor: Ensure training of personnel on this procedure.

4.0 References:

• Manufacturer’s instructions for Protein A resin (e.g., Thermo Fisher, GE Healthcare)
• ICH Q5C: Quality of Biotechnological Products
• JSA/SOP/BIO/DP/010 – Use of Nanodrop or Spectrophotometer for A280 Analysis

5.0 Definitions:

• CHO DP12: A Chinese Hamster Ovary cell line engineered to express anti-IL-8 IgG.
• Protein A: Affinity ligand that binds to the Fc region of IgG antibodies.

6.0 Materials and Equipment:

• Clarified CHO DP12 conditioned media
• Gravity flow Protein A column
• Binding buffer (PBS, pH 7.4)
• Elution buffer (Glycine-HCl, pH 3.0)
• Neutralization buffer (1M Tris, pH 9.0)
• Spectrophotometer or Nanodrop
• pH meter, sterile tubes, collection rack

SOP for Purification of Anti IL-8 Monoclonal Antibody from CHO DP12 Conditioned Media using Gravity Flow Protein A Chromatography

1.0 Objective:

To describe the procedure for the purification of anti-IL-8 monoclonal antibody (mAb) expressed in CHO DP12 cells using gravity flow Protein A affinity chromatography.

2.0 Scope:

This SOP applies to the downstream purification team involved in the capture of IgG monoclonal antibodies from clarified CHO DP12 conditioned media using gravity-based Protein A resin columns.

3.0 Responsibility:

• Downstream Manufacturing Technicians: Perform the purification procedure as outlined.

• Quality Assurance: Review and approve the documentation and ensure compliance.

• Supervisor: Ensure training of personnel on this procedure.

4.0 References:

• Manufacturer’s instructions for Protein A resin (e.g., Thermo Fisher, GE Healthcare)

• ICH Q5C: Quality of Biotechnological Products

• JSA/SOP/BIO/DP/010 – Use of Nanodrop or Spectrophotometer for A280 Analysis

5.0 Definitions:

• CHO DP12: A Chinese Hamster Ovary cell line engineered to express anti-IL-8 IgG.

• Protein A: Affinity ligand that binds to the Fc region of IgG antibodies.

6.0 Materials and Equipment:

• Clarified CHO DP12 conditioned media

• Gravity flow Protein A column

• Binding buffer (PBS, pH 7.4)

• Elution buffer (Glycine-HCl, pH 3.0)

• Neutralization buffer (1M Tris, pH 9.0)

• Spectrophotometer or Nanodrop

• pH meter, sterile tubes, collection rack

SOP for Use of MabSelect SuRe™ Column for Monoclonal Antibody Capture

1.0 Purpose:

To outline the procedure for capturing monoclonal antibodies (mAbs) using the MabSelect SuRe™ Protein A affinity chromatography column during the downstream purification process.

2.0 Scope:

Applicable to all production batches involving mAb capture using MabSelect SuRe™ column as part of the downstream processing activities at JSA Pharma Guideline biologics manufacturing facilities.

3.0 Responsibilities:

• Manufacturing personnel are responsible for the execution of the procedure.

• QA personnel are responsible for verification, compliance, and review of records.

• Engineering ensures equipment calibration and maintenance.

4.0 References:

• GE Healthcare MabSelect SuRe™ Product Manual

• JSA/SOP/BIO/DP/002 – Operation and Cleaning of Protein A Affinity Chromatography Columns

• JSA/SOP/BIO/DP/009 – Column Regeneration and Storage

5.0 Definitions:

• MabSelect SuRe™: A Protein A chromatography resin designed for high-capacity mAb capture.

• CIP: Clean-in-Place

• WFI: Water for Injection

6.0 Materials and Equipment:

• MabSelect SuRe™ column (prepacked or self-packed)

• Equilibration buffer (e.g., 20 mM sodium phosphate, pH 7.0)

• Sample containing mAb (filtered and clarified)

• Elution buffer (e.g., 0.1 M glycine-HCl, pH 3.5)

• Neutralization buffer (e.g., 1M Tris-HCl, pH 9.0)

• Wash buffer (PBS or equilibration buffer)

• Column-compatible chromatography system (e.g., ÄKTA system)

• pH and conductivity meters

• Protein quantification tools (e.g., UV detector, Nanodrop)

SOP for Ion Exchange Chromatography using Q and S Columns for Polishing of Monoclonal Antibodies

1.0 Objective

To establish a robust and validated procedure for the polishing step of monoclonal antibody (mAb) purification using ion exchange chromatography, specifically strong anion exchange (Q) and strong cation exchange (S) columns, to remove process- and product-related impurities post Protein A chromatography capture.

2.0 Scope

This SOP applies to all downstream processing personnel involved in the purification of monoclonal antibodies expressed in CHO cells, particularly for the separation of charge variants, host cell protein (HCP), residual DNA, endotoxins, and aggregates from the target protein. It is intended for use following the Protein A affinity step in both clinical and commercial GMP manufacturing environments.

3.0 Responsibility

• Downstream Processing (DSP) Operators:
  Responsible for the setup, operation, monitoring, and cleaning of ion exchange chromatography systems.
• Process Development Scientists / Engineers:
  Responsible for selecting appropriate resins, buffers, and gradients based on process characterization.
• Quality Control (QC) Laboratory:
  Responsible for performing in-process testing including pH, conductivity, protein concentration, HCP, and DNA.
• Quality Assurance (QA):
  Review batch records and ensure compliance with SOP and regulatory requirements.

4.0 References

• ICH Q6B: Specifications for Biotechnological/Biological Products
• JSA/SOP/BIO/DP/013 – Inline pH and Conductivity Monitoring
• JSA/SOP/BIO/DP/012 – Protein Quantitation by BCA and Bradford Assay
• Manufacturer’s guidelines for Q Sepharose™ Fast Flow and SP Sepharose™ Fast Flow resins
• FDA Guidance for Industry: Process Validation – General Principles and Practices

5.0 Definitions

• Q Column: A quaternary amine-functionalized resin for anion exchange, used to bind negatively charged species (acidic impurities) at near-neutral to basic pH.
• S Column: A sulfonic acid-functionalized resin for cation exchange, used to bind positively charged species (basic impurities) at acidic pH.
• Polishing Step: The purification step that removes remaining impurities to meet final purity specifications.
• Column Volume (CV): The volume occupied by the packed resin bed within a chromatography column.

SOP for Size-Exclusion Chromatography for Aggregate Removal in Final Purification

1.0 Objective:

To define the standardized procedure for performing Size-Exclusion Chromatography (SEC) to separate aggregates, fragments, and other size-based impurities from monoclonal antibody (mAb) products during the polishing step of downstream purification.

2.0 Scope:

This procedure applies to all biologics manufacturing operations where final polishing and aggregate removal is required post ion-exchange or affinity purification steps, particularly for mAbs and recombinant proteins.

3.0 Responsibility:

• Manufacturing DSP Technicians: Responsible for executing SEC setup, operation, and monitoring.
• QA Personnel: Responsible for reviewing and verifying batch records and compliance.
• QC Analysts: Responsible for in-process and final product testing (e.g., SEC-HPLC for aggregate content).

4.0 References:

• GE Healthcare, “Superdex™ 200 Increase Column Instruction Manual”
• ICH Q5C – Stability Testing of Biotechnological/Biological Products
• USP <1057> – Analytical Instrument Qualification
• JSA/SOP/BIO/DP/013 – Inline pH and Conductivity Monitoring in Chromatography Processes

5.0 Definitions:

• SEC: Size-exclusion chromatography separates biomolecules based on molecular size.
• Monomer: Intact form of mAb or protein.
• Aggregate: Dimer or multimer form of mAb, generally considered an impurity.

6.0 Materials and Equipment:

• Superdex™ 200 Increase 10/300 GL or equivalent SEC column
• Chromatography skid (e.g., ÄKTA™ Pure)
• UV detector at 280 nm
• Fraction collector
• Equilibration/Elution buffer: 20 mM Sodium Phosphate, 150 mM NaCl, pH 7.0
• pH meter and conductivity meter
• 0.22 µm syringe filters
• Protein sample (typically post-IEX or Protein A pool)

SOP for Buffer Exchange and Concentration using Tangential Flow Filtration (TFF)

1.0 Objective:

To describe the procedure for buffer exchange and concentration of monoclonal antibodies or recombinant proteins using Tangential Flow Filtration (TFF) as a part of downstream processing before formulation or storage.

2.0 Scope:

Applicable to all operations involved in the concentration and diafiltration (buffer exchange) of biologic drug substances, specifically after polishing chromatography steps and prior to sterile filtration or drug product formulation.

3.0 Responsibility:

• DSP Operators: Execute setup, operation, and cleaning of TFF system.
• QA Personnel: Ensure batch records are reviewed and deviations addressed.
• QC Analysts: Confirm sample integrity and concentration via protein assay (e.g., A280, BCA).

4.0 References:

• Manufacturer’s Instructions: Millipore Pellicon™ TFF System
• PDA Technical Report #15: TFF Applications in Biotech
• ICH Q8 – Pharmaceutical Development
• JSA/SOP/BIO/DP/012 – Protein Quantitation by BCA/Bradford Assay

5.0 Definitions:

• TFF: A membrane-based filtration technique used to concentrate and exchange buffers in bulk biomolecule solutions.
• Diafiltration: A process to replace the existing buffer with a desired formulation buffer using continuous dilution.

6.0 Materials and Equipment:

• Pellicon™ 2 cassettes with 30 kDa cutoff (or equivalent)
• TFF system with pressure gauges and peristaltic pump
• Ultrafiltration/Diafiltration buffer: e.g., 20 mM Histidine, 100 mM NaCl, pH 6.0
• Inlet and retentate bags, sterile tubing sets
• Conductivity and pH meters
• 0.22 µm filter units for sterile filtration