mAB production: Step 10 – Ultrafiltration and Diafiltration (UF/DF): Concentration and Formulation

How we concentrate the monoclonal antibody to its final strength and exchange the buffer into the stable, injectable formulation.

Welcome back to our deep‑dive series on monoclonal antibody manufacturing. In Step 9, we performed viral filtration (nanofiltration), removing any potential viruses by size exclusion. The viral filtrate is now very pure, but it is still dilute (typically 2–10 g/L mAb) and suspended in the AEX buffer (e.g., Tris or phosphate, low salt, pH 7.0–8.0).

The final drug substance for a product like Humira (adalimumab) must be:

• Concentrated to 50–150 mg/mL (typically 50–100 mg/mL for subcutaneous injection)

• Formulated in a specific buffer (e.g., citrate, histidine, polysorbate 80, trehalose) that maintains stability at 2–8°C for months

Step 10 – Ultrafiltration and Diafiltration (UF/DF) accomplishes both goals in a single, integrated operation. UF concentrates the product; DF exchanges the buffer. This is the last stop before the bulk drug substance (BDS) is released for sterile filling.

Let’s explore the principles, equipment, step‑by‑step procedure, critical parameters, and common pitfalls.

Overview: What Are Ultrafiltration and Diafiltration?

Ultrafiltration (UF): A pressure‑driven membrane process that retains molecules above a certain molecular weight cut‑off (MWCO) while allowing smaller molecules (buffer salts, water) to pass through. For mAbs (150 kDa), a 30 kDa or 50 kDa MWCO membrane is used. The retained mAb is recirculated and becomes more concentrated over time.

Diafiltration (DF): A washing process where the retentate (concentrated mAb) is continuously diluted with the new buffer (formulation buffer) while permeate (old buffer + water) is removed. This exchanges the buffer composition.

Why combine them?

• UF concentrates first (reduces volume), then DF exchanges the buffer using the same membrane.

• No need to transfer product between vessels.

• High recovery (>95–98%) when optimized.

Operating mode: Tangential flow filtration (TFF) – the feed flows parallel to the membrane surface, sweeping away concentrated product and reducing fouling. This is distinct from normal flow (dead‑end) filtration used in viral filtration.

Step 10.1: UF/DF System Components

A typical TFF system for commercial mAb manufacturing includes:

Membrane selection:

• 30 kDa MWCO – Good retention (>99.9% of mAb), lower flux, higher pressure.

• 50 kDa MWCO – Slightly higher flux, still excellent retention (>99.9%). Most common for IgG1.

• 100 kDa – Risk of mAb loss (not recommended).

Materials: Single‑use cassettes (e.g., Pall Omega, Sartorius Hydrosart, Millipore Pellicon) are common at pilot and commercial scale. Stainless steel holders are reusable.

Step 10.2: Step‑by‑Step Procedure

The UF/DF operation proceeds in three phases: concentration (UF), diafiltration (DF), and final concentration (optional). The entire process is performed at controlled temperature and transmembrane pressure (TMP).

Step 10.2.1: System Preparation and Integrity Testing

Purpose: Ensure the membrane is intact and sterile.

Process:

1. Install the UF membrane cassette in the holder.

2. Flush with WFI to remove storage solution (typically 0.1 M NaOH or 20% ethanol).

3. Perform a water permeability test (measure flux at a given TMP) to verify integrity.

4. Sanitize with 0.5 M NaOH (30 min), then flush with WFI until pH neutral.

5. Equilibrate with the starting buffer (the same as the viral filtrate) to condition the membrane.

Step 10.2.2: Ultrafiltration (Concentration)

Purpose: Reduce the volume of the viral filtrate to the target concentration (e.g., from 5 g/L to 50 g/L, a 10‑fold concentration).

Process:

1. Transfer the viral filtrate from the hold tank into the feed/retentate tank.

2. Start recirculation at low pump speed, then increase to target cross‑flow rate.

3. Apply backpressure on the retentate line to generate transmembrane pressure (TMP) .

4. As permeate is removed, the volume decreases and concentration increases.

5. Continue until the desired concentration factor (e.g., volume reduced to 1/10 of starting volume) is reached. Monitor by weight or volume.

Target parameters:

• TMP: 0.5–1.5 bar (7–22 psi). Too low → low flux; too high → membrane compaction or fouling.

• Cross‑flow rate: 3–6 L/min/m² (linear velocity ~0.1–0.3 m/s for flat sheet).

• Flux (permeate flow): 50–200 L/m²/h (LMH). Declines as concentration increases.

Monitoring: In‑line UV (A280) or refractive index can estimate concentration, but off‑line A280 measurement is more accurate.

Step 10.2.3: Diafiltration (Buffer Exchange)

Purpose: Replace the old buffer (from AEX/viral filtration) with the final formulation buffer.

Process:

1. At the end of UF, the retentate is at the target concentration (e.g., 50 g/L). Now hold that volume constant by adding formulation buffer at the same rate as permeate is removed.

2. Start the diafiltration pump to feed formulation buffer into the retentate tank at a controlled rate.

3. Maintain constant volume (tank level or weight). Permeate is removed continuously.

4. Continue for 5–10 diavolumes (a diavolume = the current retentate volume). For example, if the retentate volume is 100 L, adding 500 L of buffer (5 diavolumes) exchanges >99% of the old buffer.

How many diavolumes? The residual old buffer fraction = e^(-N) where N = number of diavolumes. For 5 diavolumes, residual = 0.67%; for 7 diavolumes, 0.09%; for 10 diavolumes, 0.0045%. Typically 5–7 diavolumes is sufficient.

Mode: Constant volume diafiltration (most common). Alternative: continuous diafiltration (add buffer continuously while concentrating – less precise).

Verification: After DF, sample the retentate and measure pH, conductivity, and osmolality to confirm they match the formulation buffer specifications.

Step 10.2.4: Final Concentration (Optional)

If the diafiltration step diluted the product (in constant‑volume DF, concentration remains the same as at the start of DF), you may need a final concentration step to reach the exact target.

Process: Stop buffer addition and continue permeate removal until the desired final concentration is achieved (e.g., 100 mg/mL). Monitor by A280.

Step 10.2.5: Recovery (Product Harvest)

Purpose: Recover the concentrated, formulated mAb from the system.

Process:

1. Stop permeate flow.

2. Use a low‑shear pump to push the retentate out of the tank through a product outlet.

3. Rinse the system (tank and membrane) with a small volume of formulation buffer (a “sweep”) to recover product remaining in the system. This is typically 0.5–1.0× the retentate volume.

4. Combine the rinse with the main pool. The final pool is the Bulk Drug Substance (BDS) .

Recovery target: >95% overall (UF + DF + rinse). Poor recovery indicates membrane binding or excessive hold‑up volume.

Step 10.2.6: Membrane Cleaning and Storage

After recovery, the membrane is cleaned and stored for reuse (if reusable) or discarded (if single‑use). For reusable cassettes:

• Flush with WFI to remove residual product.

• Clean with 0.5 M NaOH (30–60 min at 40–50°C).

• Flush with WFI until pH <8.0.

• Store in 0.1 M NaOH or 0.05% sodium azide (if compatible with next use).

Single‑use cassettes are discarded after one batch – no cleaning validation required, but higher consumable cost.

Step 10.3: Critical Process Parameters (CPPs)

Step 10.4: Equipment and Scale Considerations

Small scale (research/pilot): Bench‑top TFF systems (e.g., Sartorius Vivaflow, Pall Minimate, Cytiva ÄKTA flux) with 0.01–0.1 m² membranes.

Commercial scale (2,000–20,000 L): Large‑scale TFF skids with multiple cassettes in parallel. For a 20,000 L batch at 5 g/L starting concentration, the retentate volume after UF to 50 g/L is 2,000 L. Membrane area required: 10–40 m² depending on flux.

Key equipment features:

• Low‑shear pumps: Peristaltic or diaphragm pumps are preferred. Centrifugal pumps can be used at low tip speeds.

• Automated TMP control: PID loop controlling retentate valve based on pressure readings.

• Load cells or flow meters: For precise volume and diafiltration control.

• In‑line concentration monitor: UV (A280) or Raman for real‑time feedback.

Single‑use vs. reusable:

• Single‑use TFF assemblies (e.g., Pall Allegro, Sartorius FlexAct) are increasingly popular for clinical and commercial batches up to 2,000 L, eliminating cleaning validation.

• Reusable stainless steel systems are still common for large‑scale (>10,000 L) legacy processes.

Step 10.5: Formulation Buffer Composition – Example (Humira‑like)

For adalimumab (Humira), the final drug substance formulation is:

The DF step replaces the AEX buffer (e.g., 20 mM Tris, 50 mM NaCl, pH 7.5) with this formulation buffer.

Step 10.6: Product Quality Considerations During UF/DF

Critical monitoring: At the end of UF/DF, sample the BDS and test for:

• Protein concentration (A280, or Kjeldahl/Bradford)

• pH

• Conductivity

• Osmolality

• Aggregates (SEC‑HPLC)

• Subvisible particles (light obscuration)

• Bioburden and endotoxin

Step 10.7: What Can Go Wrong? (Troubleshooting)

Step 10.8: Post‑UF/DF – The Bulk Drug Substance (BDS)

After UF/DF and recovery, the product is now Bulk Drug Substance (BDS) . It is:

• Concentrated to the final target (e.g., 50–100 mg/mL)

• Formulated in the exact buffer for stability

• Sterile (filtered through 0.22 µm after UF/DF, or during the final fill step)

Hold and storage: BDS is typically stored in sterile, single‑use bags (e.g., Thermo HyPerforma, Sartorius Flexsafe) at 2–8°C (or −20°C for longer storage). It is held until it is released by quality control (QC) for Step 11: Sterile Filling and Finishing.

QC testing for BDS release includes:

• Identity (peptide map, or ELISA)

• Purity (SEC, CE‑SDS)

• Potency (bioassay)

• Safety (sterility, endotoxin, mycoplasma)

• General tests (pH, osmolality, protein concentration)

Only after passing all release tests can the BDS be shipped to the filling line.

What Comes Next?

The BDS is ready for its final journey: Step 11 – Sterile Filling and Finishing – where it is filtered, filled into vials or pre‑filled syringes, lyophilized (if required), inspected, labeled, and packaged. After that, the product is ready for the patient.

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