An RPLC Method with CAD for the Quantification of Residual Simethicone in Biologic Products

Foaming in bioreactors presents challenges during monoclonal antibody (mAb) manufacturing, necessitating the use of antifoaming agents such as simethicone. While effective in upstream processes, simethicone must be removed during downstream purification to meet regulatory standards, as it is considered a process-related impurity. A reversed-phase liquid chromatography–charged aerosol detector (RPLC–CAD) method was developed and validated per ICH Q2(R1) guidelines for simethicone quantification. The method demonstrated high specificity, accuracy, and sensitivity, with a limit of quantitation of 10 ppm and a linear response from 10 to 120 ppm. It was applied to evaluate simethicone clearance in an in-house IgG1 mAb process using spiking studies in scale-down chromatography models. The results showed a >4.0 log₁₀ reduction across three purification steps, confirming effective removal. This study demonstrates the suitability of the RPLC–CAD method for monitoring residual simethicone and ensuring regulatory compliance.

Therapeutic monoclonal antibodies (mAbs) are widely used to treat various human diseases, including cancer, autoimmune disorders, and chronic inflammatory conditions (1). The success of IgG mAbs has driven the development of diverse therapeutic antibody formats, such as antibody fragments, bispecific antibodies, nanobodies, and fusion proteins. During the large-scale production of these biotherapeutics in bioreactors, the oxygen demand of cells is met by sparging sterile air through the culture medium. However, sparging gas at high rates, combined with intense agitation and the presence of surface-active species like proteins at high concentrations, can lead to the formation of foam (2,3).

Foams are complex gas-liquid dispersions (with over 95% gas content) whose characteristics depend on the properties of the solution and process conditions (3). Foam formation can reduce the efficiency of gas exchange at the culture surface by creating a barrier between the culture medium and the gases in the vessel’s headspace (4). If foam formation is inadequately controlled, cells and substrates may be lost into the foam phase, reducing process productivity. In extreme cases, a “foam out” event can compromise process sterility. Controlling foam during fermentation is critical due to its adverse effects, including safety hazards. The preferred method for foam control is the use of chemical antifoaming agents (or antifoams) (5). These agents are routinely used in bioreactors to prevent foam formation by reducing the surface tension of the culture medium.

Simethicone is a highly effective antifoaming agent commonly used to mitigate the detrimental effects of foaming in cell culture media during biopharmaceutical production. It is a complex mixture of high-molecular-weight polydimethylsiloxane (PDMS) oligomers and particulate silicon dioxide (SiO₂), which enhances the defoaming properties of the silicon oil (Figure 1) (6). While simethicone offers significant advantages as an antifoaming agent in biopharmaceutical manufacturing, it is classified as a process-related impurity in the final product, according to the ICH Q6B guideline (7). Antifoaming agents and other upstream process-related impurities typically have lower molecular weights compared to biological process-related impurities such as host cell proteins (HCPs) and DNA. These smaller impurities are generally considered too small to constitute epitopes recognizable by the mammalian immune system, resulting in a relatively low immunogenicity risk (8). However, biotherapeutics manufacturers must provide evidence of process clearance of the antifoaming agent for the risk to be considered negligible.

Residual traces of simethicone are usually determined by analytical assays. As a result of the absence of chromophores in PDMS, quantitative analysis methods for simethicone include gravimetric analysis, Fourier-transform infrared (FTIR) spectroscopy, and reversed-phase liquid chromatography with an evaporative light scattering detector (RPLC–ELSD) (6,9,10). Simethicone levels can also be assessed indirectly by measuring silicon content using inductively coupled plasma mass spectrometry (ICP-MS) (8). However, each of these methods has limitations. For instance, ELSD exhibits a non-linear or sigmoidal response at low and high analyte concentrations because of concentration-dependent changes in aerosol particle size distributions (6).

In this study, we demonstrate the sensitivity of RPLC with a charged aerosol detector (CAD) for the quantitative analysis of simethicone. CAD, a mass-based detector, is particularly well-suited for detecting nonvolatile analytes regardless of their chemical properties. The detector employs nebulization to create aerosol droplets, and as the mobile phase evaporates in the drying tube, analyte particles remain and are subsequently charged in the mixing chamber. These charges are then measured by a electrometer, generating a signal proportional to the quantity of analyte present (11–13). A simplified schematic of the CAD mechanism is shown in Figure 2.

Experimental

For RPLC–CAD analysis, an U3000 HPLC system coupled with a Corona Veo RS CAD detector (both Thermo Scientific)and a 4.6 × 250 mm, 5-µm C8 column (Agilent) were used. A stainless-steel frit with a 0.5 µm pore size served as the pre-column filter. The mobile phases consisted of (A) 0.2% formic acid in 60% acetonitrile and (B) isopropanol in THF (tetrahydrofuran). The gradient program began with 0% B, ramping to 70% B within 3 min, followed by an increase to 90% B over the next 2 min, and then to 100% B in 1.5 min. A hold at 100% B was maintained for 3.5 min before returning to the initial 0% B. The total run time was 16 min at a flow rate of 1 mL/min. The column temperature was set to 50 °C, and the sample injection volume was 50 µL. The CAD was operated with an evaporation temperature of 50 °C, a power function of 1.4, and the detector flow feature activated from 6 to 12 min. The data collection rate was set at 10 Hz with a filter constant of 10 s. Data acquisition and analysis were performed using Chromeleon Chromatography Data System software, version 7.2 SR4 (Thermo Scientific).

Standard and Sample Preparations

Simethicone Standard Preparation

A stock solution of simethicone was prepared by accurately weighing USP-grade simethicone (Catalog #S1926, Spectrum Chemical) and dissolving it in THF. A series of standard solutions was then prepared by diluting the stock solution with water. For example, a 5-ppm simethicone solution was prepared to verify instrument sensitivity, while a 200-ppm solution was prepared as a stock for spiking drug substance (DS) and in-process samples. Calibration standards were prepared at concentrations of 10, 20, 40, 60, 90, and 120 ppm.

Sample Preparation

mAb1 DS and in-process samples (Protein A load and pool, Q-Sepharose Fast Flow [QSFF] load and pool, mixed-mode chromatography [MMC] load and pool, and ultrafiltration/diafiltration [UFDF] Pool) were diluted with THF and water before spiking simethicone. A 10% ADCF (animal-derived component-free) antifoam solution used in downstream clearance study was from Cytiva (PN SH30897). It was formulated by dilution of 30% simethicone emulsion using water for injection quality water.

Results

Method Qualification Results

The method was qualified according to ICH Q2R(1) guidelines for specificity, precision, linearity, accuracy, and determination of limits of detection (LOD) and quantitation (LOQ) (14).

Specificity

The specificity of the method was demonstrated by analyzing simethicone (60 ppm) alongside both mobile phases, the formulation buffer, and various in-process samples (Protein A, QSFF, MMC, and UFDF pool matrices). Figure 3 shows the overlay of chromatographic profiles for the respective samples. Simethicone eluted at 8.4 min, with no interference peaks observed within its retention time window for the mobile phases, formulation buffer, or in-process samples. The observed asymmetry of simethicone peak (symmetry factor of 0.69) is likely attributable to the nature of PDMS, which is not a single compound but a complex mixture of oligomers.

Repeatability

Repeatability was evaluated by spiking known amounts of simethicone into samples. As no simethicone was detected in the DS or in-process samples, six replicate preparations of simethicone were spiked into DS and in-process samples (MMC and UFDF pool matrices). The samples were analyzed using the RPLC–CAD method.

The results of the repeatability analysis, including retention time and peak area for individual preparations across these samples, are summarized in Table I. The retention time demonstrated excellent reproducibility, with a relative standard deviation (RSD) of less than 0.06%. However, the peak area recorded by the CAD exhibited an RSD ranging from 6.82% to 12.04% across replicates for the three sample types. The measured simethicone content ranged from 49 to 53 ppm, which aligns with the spiked concentration of 60 ppm.

Linearity

A linear relationship between analyte concentration and response was assessed to confirm the suitability of the method for its intended purpose. Linearity was determined by calculating the correlation coefficient from the regression curve.

During analysis, charged particles were detected by an electrometer, and the resulting signal was mathematically transformed into a chromatographic response (Figure 2). The raw signal current depends on the number of charges for each particle and is not inherently linear with respect to sample concentration, as it reflects the particle’s surface area rather than its volume. To enhance linearity over a broader concentration range, a built-in power function (PF) was applied (15,16).

Optimizing the PF is a critical step in the development of RPLC–CAD methods. Therefore, linearity was evaluated using PF values of 1.0, 1.2, 1.4, and 1.6. The method using PF 1.4 demonstrated excellent linearity, with a correlation coefficient of 0.9956 between simethicone concentration and the CAD response (Table II).

Limit of Detection and Limit of Quantitation

The sensitivity of the method was determined by evaluating the LOD and LOQ. The LOD represents the smallest analyte concentration that generates a measurable response, while the LOQ is the lowest analyte concentration that can be quantified with acceptable precision. LOD and LOQ were assessed by analyzing simethicone standards diluted with water to concentrations of 1, 2.5, 5, and 10 ppm. Figure 4 shows the overlay of chromatographic profiles for these diluted simethicone solutions, indicating that the simethicone peak was detectable only at concentrations of 5 ppm and above.

Six replicate preparations of the 5 ppm and 10 ppm dilutions were analyzed, and the signal-to-noise (S/N) ratios for the simethicone peak were determined. At 5 ppm, the S/N ratio ranged from 8.5 to 18.2, while at 10 ppm, it ranged from 19.4 to 44.4. Reliable quantification was achieved at 10 ppm, as the measured concentration closely matched the expected value (Table III). Based on the accuracy and precision observed at these lower ranges, the LOD was established at 5 ppm, and the LOQ was determined to be 10 ppm.

Accuracy

Accuracy was evaluated across the reportable range of the method by comparing measured results with expected values. Known amounts of simethicone were spiked into DS and in-process samples (MMC and UFDF), and their measured concentrations and recoveries were calculated. Simethicone was spiked at the LOQ level of 10 ppm and at three additional concentration levels: 20 ppm (low), 60 ppm (medium), and 100 ppm (high). Six replicate preparations were made for the medium-level spike, while three replicates were prepared for each of the other levels. Figure 5 shows an overlay of chromatographic profiles of unspiked and spiked DS samples at the LOQ, low, medium, and high levels of simethicone.

The percent recovery of the added simethicone was calculated using equation 1. Table IV summarizes the recovery of simethicone in DS across replicates at each spike level, including the mean recovery and %RSD. The recoveries for MMC and UFDF ranged from 68% to 122% and 77% to 120%, respectively.

where,

C_measured =
Measured simethicone concentration in the spiked sample

V_spike = Volume of spiked simethicone

C_spike = Concentration of the simethicone spike solution

V_sample = Volume of the sample

C_unspiked = Simethicone concentration in unspiked sample

V_total = Total volume

Simethicone Clearance Study

The removal of process-related impurities during downstream manufacturing processes is critical for managing their associated safety risks. Therefore, effective downstream processing steps that ensure the clearance of these impurities are crucial (8). During the production of an in-house IgG1 monoclonal antibody (mAb1), a 10% ADCF antifoam solution, containing approximately 3% active simethicone, was added upstream to control foaming. This antifoam was subsequently removed during downstream processing. To ensure that simethicone levels in the final DS remain within safe limits, monitoring its clearance in in-process samples is essential for risk mitigation.

The RPLC–CAD method was successfully applied to evaluate simethicone clearance during the downstream purification process of mAb1. Figure 6 illustrates the flowchart of key steps in the mAb1 downstream manufacturing process. Protein A chromatography is designed to capture mAb1 from the harvested cell culture fluid, while process-related impurities are removed in the flow-through or during subsequent wash steps. The anion exchange chromatography step (QSFF) reduces process-related impurities, such as residual DNA and HCPs, by capturing impurities while allowing mAb1 to flow through. Finally, MMC reduces product-related impurities, such as aggregates and charge variants. This step operates in a bind and gradient-elution mode for mAb1.

To evaluate simethicone clearance, systematic spiking and clearance studies were performed on qualified scale-down models of Protein A, QSFF, and MMC chromatography. The target quantity of antifoam spiked into each load was calculated for each chromatography step, and simethicone levels in the samples were quantified using a five-point calibration curve prepared with USP-grade simethicone.

To simulate a worst-case scenario for the Protein A chromatography load, the maximum allowable amount of antifoam during the 14-day cell culture process was determined to be 31.3 µg/mL (0.00313% w/w) of simethicone, the active ingredient in the 10% antifoam solution. For the Protein A chromatography step, a simethicone concentration of 0.0049% w/w was spiked, slightly exceeding the worst-case level for the upstream process. For the QSFF and MMC chromatography loads, the maximum allowable simethicone spike level was set at 450 µg/mL (0.0450% w/w) to avoid interfering with chromatography performance. The actual spike levels were 0.0123% and 0.0173% w/w for QSFF and MMC samples, respectively.

The load and pool fractions from each chromatography step were analyzed using the RPLC–CAD method to quantify simethicone. The results of the spiking and clearance studies for the three chromatography operations are summarized in Table V. The measured simethicone concentration in the Protein A load was 49 ppm, while the concentration in the Protein A pool was below the LOQ (<10 ppm) (Table V and Figure 7). The results for QSFF and MMC chromatography are also presented in Table V. The residual levels of simethicone in the pool fractions were determined to be significantly lower than their starting levels in the load.

The clearance factor for the impurity was expressed as the log10 reduction value (LRV), calculated using the following equation:

where,

LRV = Log Reduction Value

C_load = Impurity concentration in the load material

V_load = Volume of the load material

C_pool =
Impurity concentration in the pooled (eluted) material

V_pool = Volume of the pooled (eluted) material

Simethicone removal by each chromatography step was determined based on the LRV, averaged across duplicate runs. The total process LRV was calculated as the sum of the average LRV for each purification step. An average LRV below 1.0 is considered insignificant and is not reported as clearance. The results demonstrate that the mAb1 purification process achieved a total simethicone clearance exceeding 4.0 LRV (Table VI).

Conclusion

An RPLC–CAD method was developed to quantify residual simethicone in mAb drug substances and in-process samples. The method was qualified in accordance with ICH guidelines for specificity, repeatability, accuracy, linearity, and LOD and LOQ. The method’s LOD and LOQ were estimated to be 5 ppm and 10 ppm, respectively. This method was successfully applied to an in-house IgG1 mAb1 antifoam clearance study. Data from systematic spike-and-clearance studies demonstrated that the downstream process achieves robust clearance of simethicone, a process-related impurity. This study highlights that RPLC–CAD is a sensitive and reliable quantitative method for monitoring antifoam levels in the mAb manufacturing process.

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