Physical Property Evaluation Techniques in Biopharmaceutical Development: Manufacturing Process

by Malvern Panalytical, Tuesday, 17th August 2021

This page provides guidance on the items to be evaluated at each stage of biopharmaceutical development and the evaluation techniques available from Malvern Panalytical. Here, we introduce in detail the “manufacturing process.”

Once the formulation conditions are determined, the next step is scaling up. As the manufacturing process progresses, submicron aggregates that are insoluble and not visible in small scales (100-1000 nm SVP) may become apparent. Reports are increasing that SVP is associated with an increased risk of immunogenicity. It is believed that the patient’s immune system may recognize protein aggregates as infectious particles, such as viruses, causing an undesirable immune response. Malvern Panalytical’s measurement techniques support SVP evaluation during scale-up.

Assessment of Size Changes by Average Particle Size and PdI (DLS・SLS)

Comparison of Antibody Size Changes Before and After the Manufacturing Process

Proteins are composed of polypeptide chains that are sensitive to various processing conditions, such as preparation methods, storage methods, and buffers, so it’s important to understand the physical property changes in the manufacturing process. By using Dynamic Light Scattering (DLS) and Static Light Scattering (SLS), aggregation, and fragmentation can be evaluated. The following figure shows the DLS results of untreated and treated therapeutic antibodies. The horizontal axis represents size, and the vertical axis represents light intensity distribution (Intensity (%)). The average particle size of the untreated antibody sample (blue) was about 11 nm, while the treated (red) sample had an average particle size of about 50 nm, confirming the presence of aggregates. These results suggest that changes occur in the antibody samples before and after treatment. Additionally, the untreated antibody sample also shows a broad distribution, with a Polydispersity Index (PdI) exceeding 0.1 at 0.14, suggesting the presence of small antibody fragments. These results coincide with the SLS-determined absolute molecular weight of both therapeutic antibodies shown in the table, which were slightly less than the theoretical value of 145 kDa.

By monitoring these size changes in the product development process using DLS and SLS, aggregation and fragmentation can be confirmed.

Table: Static Light Scattering Measurement Results

*Ideal molecular weight of the antibody sample is 145 kDa
→By using DLS and SLS in combination, it is possible to more closely monitor aggregation and fragmentation during the manufacturing process.

Formulation Optimization Using Average Particle Size (DLS)

Temporal Observation of Size Changes in Drug-Loaded Microemulsions

The size characteristics of microemulsions are essential for ensuring safe and effective administration. By monitoring changes in size distribution with DLS, valuable information can be gathered for formulation optimization. The upper section shows the effect of drug uptake into microemulsions. The horizontal axis is size, and the vertical axis is light intensity distribution (Intensity (%)). Red represents only the microemulsion, and green represents the microemulsion + drug results. It can be confirmed that the main peak of the drug-free tens of nm shifts to the right with drug incorporation, increasing in size. The peak around 2 μm represents the insoluble drug leached from the microemulsion. The lower section monitors the mean size of drug-loaded microemulsions over time after dilution. The vertical axis is the Z-average particle size. If no drug is leached, the average size should remain constant; thus, an increase in size is linked with drug leaching.

By following the changes in the mean size of microemulsions in this way, it is also possible to check the proportion of leaching drugs.

Thus, by monitoring size changes in microemulsions using DLS, formulation optimization becomes possible.

→Supporting formulation optimization by monitoring size changes in microemulsions using particle size distribution and average particle size as indicators

SVP Concentration Quantification by High Concentration Using Scattered Light (NTA)

SVP Detection by High Concentration of BSA

In the late stages of the manufacturing process, protein concentrations become high, so it is important to evaluate dispersion stability under this condition.

Nano Particle Tracking Analysis (NTA) enables the detection of sub-micron and nanosize particles invisible in a light microscope in liquid, making it suitable for SVP concentration quantification.

The figure below analyzes SVP contained in BSA solutions of varying concentrations under the same formulation using NTA. Comparing the particle size distribution for each BSA concentration shows that at 50 mg/mL (blue), particles over 400 nm are almost undetected, and the proportion of particles over 400 nm increases as concentration increases.

Using NTA, it is possible to measure changes in SVP concentration that occur due to sample concentration.

→Visual SVP verification and quantification under high concentration is possible

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