Clarifying Complex Protein Samples with Faster Analysis

Assessing protein samples by mass photometry and size exclusion chromatography

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TECHNICAL NOTE

Assessing protein samples by mass photometry and size exclusion chromatography

Mass photometry is an analytical tool that enables the accurate mass measurement of single molecules in solution, in their native state and without the need for labels. In this technical note, mass photometry is compared to the industry's gold standard, size exclusion chromatography (SEC), for the analysis of protein abundance and antibody aggregation.

Liesa Verscheure1, Jelle De Vos1, Wiktoria Sadowska2, Thomas Martens1, Weston Struwe2, Pat Sandra1, Justin Benesch2, Koen Sandra1 1RIC biologics, President Kennedypark 6, 8500 Kortrijk, Belgium

2Kavli Institute for Nanoscience Discovery, Dorothy Crowfoot Hodgkin Building, University of Oxford, OX1 3QU, UK

Mass photometry and size exclusion chromatography (SEC) are complementary analytical tools that can provide users with a wealth of information regarding the biochemical and biophysical properties of biomolecules within a sample. However, when comparing datasets captured by the two techniques, there are some important considerations to be aware of.

In this technical note, these considerations are explored. Mass photometry and SEC data are compared in two case studies. The first case study explores how the two techniques can be used to quantify the relative abundance of each protein in a sample mixture, across a wide mass range, while the second focuses on how they can characterize antibody aggregation.

Case study 1: Assessing protein abundance within a sample mixture

Differences between SEC and mass photometry are illustrated by analyses of a sample mixture consisting of four different proteins (Fig. 1). The analyses, which used SEC-UV and mass photometry, were done by the laboratory of Professor Justin Benesch (University of Oxford).

Although the same sample mix was used for both the SEC- UV and mass photometry measurements, there is a clear disagreement as to which proteins are the most abundant within the mixture: SEC-UV suggests thyroglobulin and ferritin are the most abundant, whereas mass photometry suggests it is conalbumin and aldolase. To understand this apparent discrepancy, one must first consider the fundamental principles underlying how each technique works.

Fundamental principles of mass photometry vs. SEC

Mass photometry, as a single molecule technique, provides a particle count versus mass, i.e., it detects and counts the number of particles of a given mass. Consequently, the intensity of a mass photometry peak (the area under the peak) corresponds simply to the absolute number of molecules with the given mass that were detected during the measurement, which is proportional to the molecular concentration.

By contrast, SEC-UV analysis measures UV absorbance vs. column elution time. This data can be converted to absorbance vs. mass using the species' molecular weights (if known) and the fact that the species of greatest hydrodynamic volume usually elutes first. However, the absorbance data is not only a function of the molecule's concentration; it also depends on its UV-absorbing properties. The specifications of the UV detector itself, e.g., its sensitivity at a particular UV wavelength, also influence the data.

However, when the molar extinction coefficient of each molecule is known, the concentration of each molecule within a mixed sample can be determined from the absorbance data using the Beer-Lambert law.

Mass photometry agrees with normalized SEC-UV analysis

Applying the above approach to each protein in the sample gives rise to a normalized SEC-UV data profile that visually appears to match the mass photometry data (Fig. 1). Furthermore, quantification of the relative abundance of each protein confirms that the results of SEC-UV and mass photometry are in very close agreement (Fig. 2).

Fig. 1 Analysis of the same sample by mass photometry and SEC-UV illustrates fundamental differences between the techniques. The sample analyzed contained a mixture of four proteins: conalbumin, aldolase, ferritin and thyroglobulin. The molecular weights of each protein within the mixture were known and used to convert the SEC-UV profile from absorbance vs. elution time to absorbance vs. mass. The molar extinction coefficients of each protein were used to normalize the absorbance data.

Fig. 2 Abundance (%) of each protein within a sample mixture as determined by mass photometry and SEC-UV. For mass photometry, the number of counts for each molecule is expressed as a percentage of the total number of counts. For SEC-UV, the relative abundances of each molecule are shown, before (SEC-UV) and after (SEC-UV normalized) normalizing for the molar extinction coefficient.

Experimental details

• The SEC measurements were performed on an Agilent 1260 Infinity II with a Superdex 200 increase 3.2/300 column, operated in accordance with the manufacturer's recommended guidelines
• A 10 µL volume of the sample mixture was loaded on to the SEC column (the concentration of each protein in the mixture was 14 µM except for ferritin at 1.4 µM)
• The same sample mixture was used for mass photometry measurements, but the mix was diluted 1000-fold prior to measurement, to ensure that the concentration was within the appropriate range for this technique

Case study 2: Monitoring aggregation levels of monoclonal antibodies

Aggregation is an important quality attribute of many protein- based biopharmaceuticals, including monoclonal antibodies (mAbs) and multi-specific antibodies, which can influence product efficacy and safety. Monitoring of aggregation levels is therefore essential.

SEC is widely considered to be the gold-standard analytical tool for assessing nanometer-sized aggregates and is often combined with multi-angle light scattering (MALS) to enable the determination of molecular weight and size. However, the technique can be complicated by several factors, including column and mobile phase optimization.

In contrast, mass photometry detects light scattered by single molecules, enabling the measurement of the molecular mass of biomolecules, in solution, and this technique can analyze small (µL) sample volumes at low concentrations (100 pM up to 100 nM) under native conditions within a few minutes.

In the presented analysis, carried out by RIC biologics (Kortrijk, Belgium), both SEC and mass photometry were used to measure aggregation levels of trastuzumab, a monoclonal antibody, and several trastuzumab biosimilars.

Trastuzumab is a humanized IgG1 monoclonal antibody that can inhibit HER2 signalling pathways as well as activate antibody- dependent cell-mediated cytotoxicity, helping to facilitate the treatment of cancers that overexpress the HER2 cell surface receptor. In recent years, trastuzumab biosimilars have been developed and chromatography has been at the forefront of assessing critical quality attributes, including aggregation, during biosimilar development.

The results of this case study demonstrate that SEC and mass photometry are complementary, highlighting the usefulness of mass photometry as an orthogonal technique for monitoring aggregation of biopharmaceuticals such as mAbs.

Experimental methods

SEC and SEC-MALS measurements were performed on an Agilent Technologies 1260 Bio-inert HPLC Infinity II system equipped with a diode-array detector (DAD) and coupled to a refractive index (RI) detector and a Wyatt miniDAWN multi- angle light scattering detector. Sample compounds were first separated according to their hydrodynamic radius under native conditions using a SEC column and detected using a DAD, MALS detector, and RI detector consecutively. By using the DAD or RI signals as a concentration source, the light scattering data from multiple detector angles can be used to determine the molecular weight (MW) of analyte peaks. The sample load was increased to 315 µg to allow for accurate MW determination of the protein aggregates.

Mass photometry data was acquired using a TwoMP system, measuring the interference between the scattered light coming from individual sample molecules and the reflected light of the glass slide measurement surface. The resulting signal (interferometric contrast) is directly correlated with molecular mass and can thus be easily converted using protein standards of known MW. A detailed overview of the instrumentation and experimental conditions are provided in Tables 1 and 2 below.

Table 1 Experimental conditions for SEC-MALS

Table 2 Experimental conditions for mass photometry

Interpretation of SEC data

Trastuzumab-producing Chinese hamster ovary cell (CHO) clone supernatants samples were purified using Protein A affinity chromatography and analysed using SEC.

Fig. 3 shows the SEC chromatograms of the trastuzumab originator (Herceptin®, i.e., the 'parent molecule' or reference product) and four selected CHO clones. Distinct differences in both the high MW area and the low MW area, respectively left and right of the main monomer peak, can be distinguished. The main high MW peak is associated with a dimer of trastuzumab, which is particularly pronounced in clones 3 and 10.

The relative peak areas for both monomer and dimer species (obtained from the SEC-UV chromatogram), which are the main molecules of interest, are provided in Table 3.

To derive an estimation of the MW, a SEC-MALS experiment was run for Herceptin® and CHO clone 10 (Fig. 4). The latter showed a MW of 146.5 kDa for the monomer and 295.5 kDa for the dimer, which agrees well with the MW of the concurring peaks of the originator.

Denaturing SEC-mass spectrometry analysis showed that noncovalent dimers are present in CHO clone 10, whereas covalently bound dimers are found in the originator product1. This also explains the retention time difference between dimer peaks observed in the clones versus the originator.

Fig. 3 SEC-UV chromatograms of trastuzumab originator (Herceptin®) and trastuzumab-producing CHO clones (UV 280 nm).

Fig. 4 Molecular weight determination by SEC-MALS of monomer and dimer peak for trastuzumab originator (Herceptin®) and trastuzumab-producing CHO clone 10 (dRI: differential refractive index, LS: light scattering, MW: molecular weight).

Trastuzumab sample analysis by mass photometry

Each of the samples were also analyzed by mass photometry and measured at a final concentration of 10 nM (Fig. 5). By using albumin, α-mannosidase and thyroglobulin as calibrants, the MW could be directly interpolated from the data. The MW of the trastuzumab monomer was measured as 155 kDa and for the dimer, 300 kDa, both of which are consistent with the values determined by SEC-MALS. For the analysis of all mass photometry data sets, and to ensure consistency, monomer and dimer peaks are defined as the 120 -190 kDa and 280 - 350 kDa mass intervals, respectively. Percentage abundance of each species is calculated by determining the number of counts (i.e., those within the relevant defined mass interval) as a proportion of the total count number.

Fig. 5 Mass photometry mass histogram of trastuzumab originator (Herceptin®) and trastuzumab-producing CHO clones. Monomer peaks are highlighted in blue, dimer peaks in orange.

Normalizing SEC-UV data

When comparing % abundance for both monomer and dimer from SEC-UV (Table 3) and mass photometry (Table 5), we can see that the data is broadly in agreement, with clones 3 and 10 containing the greatest abundance of dimers relative to the predominant monomeric species. However, for a true quantitative comparison, a correction step is necessary, as previously discussed. For biotherapeutic proteins such as trastuzumab, the DAD detector response at 280 nm is predominantly related to the number of tyrosine and tryptophan residues it contains. When dimers are formed, a higher number of absorbing residues are present per molecule with respect to the monomer, which affects the peak height (detector response) and consequently also the peak area. The Beer-Lambert law was used to convert the measured peak heights in the SEC chromatogram to analyte concentration [g/L], also applying a correction factor for the UV flow cell. The concentration was subsequently converted into molar concentration [mol/L] by using the MW of the monomer and dimer species, respectively. An overview of the monomer and dimer abundance for all samples, after normalizing absorbance for molar extinction coefficient is reported in Table 4.

Table 3 Monomer and dimer abundance from SEC-UV chromatograms. To calculate percentage abundance of both monomer and dimer, the individual peak area for each was calculated and expressed as a percentage of the total/combined peak area.

Table 4 Monomer and dimer abundance after normalizing SEC-UV absorbance data

Table 5 Monomer and dimer abundance determined by mass photometry. The 120 -190 kDa mass interval defines the monomer peak and the 280 - 350 kDa mass interval, the dimer peak. Percentage abundance of each species is expressed as a proportion of the total number of counts

Conclusion

Analysis of protein aggregates in biopharmaceuticals is a prerequisite to assure product safety and efficacy. SEC, a gold standard technique, allows for a robust determination of protein aggregation, where the molecular separation relies on the difference in hydrodynamic radius. When coupled to a MALS detector, the analytical technique can identify both the size and molecular weight of the analytes within a 30-minute analysis run time.

Mass photometry is an analytical method that can analyze samples under native conditions within a one-minute analysis run time by interpreting the light scattering data from individual analyte molecules approaching the glass slide interface. The underlying detection principle of each of these analytical methods is fundamentally different and there are considerations to be made when comparing data from both, particularly when quantitatively assessing the relative abundance of different molecules in a sample mixture.

As outlined in this technical note, to account for this, the measured peak area absorbance values should be normalized by converting to molar concentration, thereby allowing an unbiased comparison of samples analyzed by SEC and mass photometry. This was illustrated in Case Study 1 with a simple protein mix and in Case Study 2 with several trasuzumab biosimilars.

SEC and mass photometry provided comparable results in both cases; any remaining differences in the data captured by both techniques likely relate to the parameters/criteria used for peak integration. Where the trastuzumab biosimilars are concerned, if the monomer-dimer ratio is concentration dependent, the differences in the concentration of antibody sample used for SEC versus mass photometry would also have an impact upon the relative abundance of monomer versus dimer.

Biopharmaceuticals such as monoclonal antibodies are complex and the requirement to ensure efficacy and safety often necessitates the use of several analytical tools to provide a comprehensive view. As shown in this technical note, mass photometry and SEC data - for a simple protein mixture, as well as several trastuzumab biosimilars - are in agreement, confirming the validity and utility of mass photometry as an orthogonal analytical technique.

References

1 Liesa Verscheure, Gerd Vanhoenacker, Sonja Schneider, Tom Merchiers, Julie Storms, Pat Sandra, Frederic Lynen, and Koen Sandra, Analytical Chemistry 2022 94 (17), 6502-6511

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