Performing mass spectrometry analysis on minimally purified bioreactor antibody samples using in-line protein A purification
Ronan Gough1, Vicky Smith1, Stephen Lock2 and Sibylle Heidelberger2
1CPI, UK; 2SCIEX, UK
Introduction
Monoclonal antibodies (mAbs) are either produced from cell cultures in batches or in a continuous manufacturing environment. They are the most successful biotherapeutics used for treating autoimmune diseases and various types of cancer.1 During their production, samples are often taken to assess protein production and monitor the process using process analytical technology (PAT) methods.2 Ideally, PAT should provide a rapid analysis and measurement of critical process parameters that affect critical quality attributes (CQAs).2 Mass spectrometry (MS) has been commonly applied directly to purified mAb samples but crude bioreactor samples usually require extensive and time-consuming clean-up before they can be analyzed by MS.3
The molecular mass of mAbs or intact proteins can be easily measured using MS, which is a straightforward and widely adopted method. This test can provide information about the identity of the protein and its glycosylation profile, which is a CQA. While it is common to measure the glycan profile at the end of the culture process, monitoring it during production in the bioreactors can enable feedback process control for mAb quality.3
Abstract
In this study we have incorporated an in-line protein A purification step into a liquid chromatography–mass spectrometry (LC-MS) workflow. After a simple sample filtration, the sample underwent a rapid analysis and quantification with minimal sample preparation. The method provides CQA information and protein titer in under 45 minutes after reactor sampling.
Figure 1. IgG sample was taken from the bioreactor, cell debris was removed, and the LC-MS system was run with in-line purification. A) LCUV chromatogram for IgG sample B) An extracted ion chromatogram (XIC) centred at m/z 3293.4 amu (The most abundant charged state of the IgG sample with an extraction window of ± 0.25 amu) of the same sample. C) Raw MS spectrum retention time 13.6 to 14.2 min with a mass range of m/z 2200 to 5000 Da..
Key features of in-line protein A purification for LC-MS analysis
- Direct analysis of bioreactor samples: Analyze bioreactor samples by MS with minimal sample preparation
- Confident identification: Intact mass analysis and glycan profile for Immunoglobulin G (IgG) antibodies
- Accurate quantitation: Quantitation by UV and LC-MS at concentrations as low as 0.1 mg/mL
Methods
Sample preparation: Bioreactor samples (950 µL from a batch process) were centrifuged (1000 g, 10 minutes) to remove cell debris.
Chromatography: Samples were separated using an ExionLC system that included a pair of ExionLC AD pumps (P/N: 5036653), an ExionLC AC pump (P/N: 5036649) and an ExionLC UV detector (P/N: 5036652). The column was a Bio-monolith protein A column (5.2 mm x 4.95 mm, Agilent, P/N: 5069-3639) held at 24°C.
The 10 µL centrifuged bioreactor samples were loaded onto the protein A column using a mobile phase consisting of 20% acetonitrile in 50mM Tris HCl (pH 8). The flow rate was set to 0.05 mL/min. The flow through was directed into a waste line using an inbuilt 6-port switching valve on the MS system. After 5 minutes, the flow rate was increased to 0.4 mL/min for 5 minutes to remove any non-specifically bound material.
The valve position was then switched to direct the flow into the MS. The retained IgG was released from the protein A column using a lower-pH eluent (20% acetonitrile, 80% water containing 0.1% formic acid). The bound non-IgG material on the column was subsequently cleaned off using a mobile phase solution that contained 95% acetonitrile, 5 % water containing 0.1% formic acid. The valve was then switched back to the load position. The total runtime was 24 minutes.
Mass spectrometry and UV detection: Peaks were detected at a UV wavelength of 280 nm (sampling rate of 10 Hz) and by MS detection.
The mass spectrometer was an X500B QTOF system (SCIEX). The system was calibrated with ESI positive calibration solution X500B (P/N: 5049910). The TOF calibration gave a mass accuracy of less than ±0.7 ppm for MS and of less than ±0.5 ppm for MS/MS. Full scan TOF spectra were acquired for a mass range of m/z 900 to 5000 with an accumulation time 0.5 s using an ion spray voltage of 5500 V and collision energy of 10 V. The ion source gases 1 and 2 were set to 50 psi, curtain gas was set to 35 psi, the CAD gas was set to 7 psi and the temperature was 500°C.
Data processing: Data collection and analysis were performed using SCIEX OS software, version 2.2 with Bio Tool Kit micro-application installed.
Results and discussion
Figures 1 and 2 show that the IgG present in bioreactor samples can be identified and confirmed using the Bio Tool Kit micro-application in SCIEX OS software. The IgG eluted at the same time in both the standard and bioreactor samples. However, the bioreactor sample had a slightly different glycan isoform pattern with more of the G1F isoform present, compared to the standard used for the external calibration line.
Figure 2. Chromatograms and mass spectra for an in-house IgG standard and depth-filtered bioreactor sample. Chromatograms of an in-house IgG standard and from a depth-filtered bioreactor sample diluted 1:2 (v/v) with water are shown in panels A and B, respectively. Raw mass spectra from the highlighted regions in panels A and B are shown in panels C and D, respectively (RT 13.6 to 14.42 min). The expanded region of these spectra are shown in panels E and F, respectively.
Figure 3 shows overlaid traces from a standard (blue) and a blank injection performed after a standard injection (pink). In both the UV and MS traces, no carryover was observed. The UV trace shows that the protein eluted 0.5 minutes before the MS peak due to the dead volume between the UV cell and the MS source, which is after and in-line with the UV detector. Figure 4 shows the calibration curves obtained for both UV and MS by injecting calibration standards. The chosen range covered only 1 order of magnitude; however, this is typical of the range monitored using other techniques in process control. Both assays were linear across this range with R2 values >0.98. The UV calibration curve (Figure 4) was used to calculate the 0.54 mg/mL concentration of IgG from the bioreactor sample shown in Figure 2.
Throughout the method development and testing, the column performance remained consistent. No noticeable changes were observed during the analysis, which consisted of more than 70 injections.
Figure 3. Overlaid chromatograms for a standard and blank injection. The 0.25 mg/mL standard and blank injections are shown in blue and pink, respectively. The UV trace is shown at the top and an XIC (centered at m/z 3293.4 with an extraction window of ± 0.25 amu) is shown at the bottom.
Figure 4. Standard curves for concentration versus UV peak area (top) or concentration versus peak area of an XIC at m/z 3293.4 with an extraction window of ±0.25 (bottom)
Conclusion
- In-line protein A purification in the LC-MS method allows for sample analysis directly from a bioreactor, enabling cell removal and IgG detection by MS without any further preparation
- Minimization of non-specific binding to the protein A column was achieved by including 20% acetonitrile in the loading mobile phase and incorporating a high flow rate wash step
- Using available samples, a linear standard curve allowed quantitation of IgG at concentrations as low as 0.1 mg/mL in bioreactor samples
References
- Lu R. et al. (2020), Development of therapeutic antibodies for the treatment of diseases, Journal of Biomedical Science, Volume: 27, Article number: 1.
- Chopda V. et al. (2021), Recent advances in integrated process analytical techniques, modeling, and control strategies to enable continuous biomanufacturing of monoclonal antibodies. Journal of Chemical Technology & Biotechnology Volume 97, Issue 9, Pages 2317-2335.
- Rathore D. et al. (2018), The role of mass spectrometry in the characterization of biologic protein products, Expert Review of Proteomics, Volume: 15, Issue: 5, Pages: 431-449.