Exosomes Part II: Clarification and Ultrafiltration Process Breakdown

Clarification Filtration: Sample Pretreatment and Standardization

1. Sample Types and Pretreatment Plans

To accommodate the processing needs of exosomes from different sources, the clarification strategies are summarized in Table 5 below:

Table 5. Clarification Process Plan and Recommendations

2. Standardized Pmax Operating Procedure

1. System Assembly and Parameter Setup

Device Setup:

Assemble the filtration system according to Pmax requirements (pump → pressure sensor → filter → collection tank). Prefer depth filter cassettes (e.g., Cobetter 1070PE) or PP capsule filters.

Flux Matching:

Maintain flux at 150–200 LMH for adherent cell samples; 100–150 LMH for suspension cells, plant, or milk-derived samples.

Hold-Up Volume Measurement:

Pre-fill the tubing with deionized water, set initial flux at 150 LMH, purge air, and measure hold-up volume (15–45 mL) for subsequent yield calibration.

2. Process Control During Filtration

The Pmax operating steps are summarized in Table 6 below:

Table 6. Clarification Operation Steps

3. Data Analysis and Scale-Up Calculation

Resistance–Load Curve:

Plot filtration load (L/m²) on the X-axis and filtration resistance (psi) on the Y-axis to generate the data trend (see Figure 1).

Safety Factor and Installation Area:

Recommended installation area: A^recommend= A^min × 1.5.

The actual installation area (A^install) should be ≥ A^recommend, and the safety factor can be calculated as SF = A^install / A^min.

Ultrafiltration and Buffer Exchange: Process Adaptability Testing

1. Hollow Fiber Selection

Exosomes are flexible and deformable nanoscale vesicles. Under external forces such as transmembrane pressure (TMP) or shear stress, they can temporarily change shape—flattening or stretching under pressure. Factors like pressure level and buffer properties (including viscosity, ionic strength, and osmotic pressure) can influence this deformation. As a result, exosomes may appear smaller in size and pass through membrane pores that are smaller than their nominal diameter. Therefore, experimental validation is essential when selecting hollow fiber pore sizes and optimizing process parameters.

Based on application data, the recommended hollow fiber configurations for exosome ultrafiltration are summarized in the table below:

Table 7. Ultrafiltration Selection

2. Determining the Optimal Shear Rate

Objective:

Identify the shear rate range that maintains high flux while preserving exosome stability.

Shear Rate Settings:

Set tangential flow rates corresponding to shear rates of 2000 s⁻¹, 3000 s⁻¹, and 4000 s⁻¹, keeping transmembrane pressure (TMP) constant (initially recommended at 3–7 psi).

Monitoring Parameters:

Average flux (LMH): Record permeate flow per unit membrane area.
Process time: Measure the time required to reach the same concentration factor.

Exosome Stability:

Particle size distribution: Use NTA or DLS to compare average particle size and polydispersity index (PDI) before and after processing.
Marker integrity: Detect marker proteins such as CD63 and TSG101 by Western blot.
Morphology: Examine exosome structure by cryo-electron microscopy (Cryo-EM) to check for rupture or aggregation.

Conclusion:

Select the shear rate that achieves high flux, short processing time, and stable exosome characteristics (PDI < 0.2 and marker recovery > 90%).

3. Determining the Optimal TMP

Objective:

Identify the TMP range that maximizes flux while preventing exosome leakage through the membrane.

Experimental Design:

TMP gradient settings:
Based on feed viscosity differences (e.g., high-viscosity milk-derived exosomes vs. low-viscosity MSC-derived exosomes), set TMP gradients as follows:
① Low-viscosity feed: 3 psi, 5 psi, 7 psi.
② High-viscosity feed: 10 psi, 15 psi, 20 psi.

Monitoring parameters:

Flux–TMP curve: Plot the relationship between flux and TMP to identify the inflection point where flux becomes pressure-independent.

Exosome leakage:

① Measure exosome content in permeate (using NTA or ELISA-based marker quantification).
② Leakage threshold: <5% (based on initial total particle count).

Concentration polarization assessment:

Analyze the correlation between flux decline rate and TMP.

Conclusion:

Select the TMP value just before the inflection point (where flux is still pressure-dependent) and ensure no significant exosome leakage.

4. Determining the Optimal Concentration and Diafiltration Factors

Objective:

Balance flux, processing time, impurity removal efficiency, and exosome activity.

Experimental Design:

Concentration factor optimization:
Set target concentration factors at 5×, 10×, and 15× (consider potential osmotic pressure effect during high concentration diafiltration).

Monitoring parameters:

① Exosome recovery rate (NTA or ELISA).
② Residual impurities (total protein concentration via BCA assay).
③ Aggregation risk (changes in PDI).

Diafiltration factor optimization:

Use equal-volume diafiltration (DF) mode, with dialysis multiple ratios of 3×, 5×, and 7× (buffer volume multiple).

Monitoring parameters:

① Impurity removal efficiency (HCP content, pH, or conductivity changes).
② Exosome recovery and activity (marker expression levels).

Conclusion:

Select the combination of concentration and diafiltration parameters that yields >90% exosome recovery and >95% impurity removal.

5. Hollow Fiber Operation Procedure

Ultrafiltration and buffer exchange workflow:

Module installation → Water rinse (purified water) → 0.5 mol/L NaOH sanitization → Water rinse (purified water) → Water flux test → Integrity test → Buffer equilibration → TMP optimization → Concentration and diafiltration (monitor conductivity/pH during diafiltration) → Product recovery (top flush) → Buffer rinse → Water rinse → 0.5 mol/L NaOH cleaning → Water rinse → Water flux test → Integrity test → Storage in 0.1 mol/L NaOH.

(1) System Assembly and Pretreatment:

Install the hollow fiber cartridge (selected according to MWCO), and connect the pump, pressure sensors, and feed/retentate reservoirs. Flush the system with ultrapure water until air is completely removed. Perform alkaline sanitization with 0.5 mol/L NaOH, followed by a thorough water rinse.
Before use, measure water flux and perform an integrity test. Equilibrate the membrane with buffer for 3–5 minutes before sample loading

(2) Parameter Initialization:

Set the initial shear rate (e.g., 3000 s⁻¹) and TMP (e.g., 0.8 bar).
Start tangential flow recirculation and allow the system to stabilize for 3–5 minutes.

(3) Concentration Phase:

Adjust the retentate-side valve to control TMP.
Continuously monitor permeate flux, pressure, and feed volume.
Record process time and operational parameters throughout the run.

(4) Diafiltration Phase:

Switch to equal-volume diafiltration mode.
Add diafiltration buffer at a constant rate while maintaining system pressure.
Monitor conductivity until stable — indicating completion of impurity exchange.

(5) Product Collection and Cleaning:

Collect the retentate (exosome product) and analyze recovery rate and residual impurities.
Clean the hollow fiber filter cartridge with 0.5 mol/L NaOH, then rinse thoroughly with ultrapure water. Recheck post-use water flux to evaluate cleaning effectiveness and perform another integrity test to confirm that the hollow fiber remains intact. For storage, soak the used hollow fiber filter in 0.1 mol/L NaOH solution as the preservation medium.

Quality Control

1. Physical Characterization

Particle Size and Concentration:

① Method: Nanoparticle Tracking Analysis (NTA) or Dynamic Light Scattering (DLS).
② Specification: Particle size distribution between 30–150 nm with a PDI < 0.2.
Morphology: Evaluated by Transmission Electron Microscopy (TEM) to confirm vesicle integrity.

2. Biochemical Characterization

Marker Identification:
① Positive markers: CD9, CD63, and CD81 (assessed by ELISA or Western blot).
② Negative markers: Calnexin and Cytochrome C (used to exclude cellular debris contamination).

Nucleic Acid Content:
① RNA: qPCR analysis for miRNA or total RNA quantification (RIN > 7).
② DNA: Residual genomic DNA content should be < 5%.

3. Safety Evaluation

Endotoxin: Measured by the Limulus Amebocyte Lysate (LAL) assay; pharmaceutical-grade samples must be < 0.25 EU/mL.
Sterility: Tested by membrane filtration in accordance with pharmacopeial microbial limits.
Host Cell Protein (HCP) Residue: Quantified by BCA assay or ELISA, with threshold values defined by sample origin.

4. Functional Verification

Bioactivity:

①Cellular Uptake Assay (Fluorescently labeled exosomes analyzed by flow cytometry or confocal microscopy).

② In Vitro Functional Assays (For example, assessing migration-promoting or anti-inflammatory effects of stem cell–derived exosomes). Drug Loading Efficiency (if applicable): Determined by HPLC or UPLC for encapsulation rate and loading capacity.

Exosomes are an emerging type of biological carrier. Their production process and quality control are still being refined to support applications in biomedicine and aesthetic medicine. Isolation, purification, and quality control are key steps for both research and practical use. 

With ongoing technological progress, exosome industrialization will advance rapidly. In the pharmaceutical field, standardized systems for production and quality control will help ensure their clinical safety and effectiveness.