The importance of being metal-free: The critical choice of column hardware for size exclusion chromatography coupled to high resolution mass spectrometry

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Article

Reference

The importance of being metal-free: The critical choice of column hardware for size exclusion chromatography coupled to high

resolution mass spectrometry

MURISIER, Amarande, et al .

Abstract

The goal of the study was to evaluate the possibilities offered by a new generation of metal-free SEC column to perform direct SEC-MS of protein biopharmaceuticals using ammonium acetate as the main mobile phase additive. The prototype metal-free SEC column hardware used in this work was a polyether ether ketone (PEEK) infused stainless steel tube including PEEK frits. This PEEK-lined column provides a fully bioinert and metal-free fluidic path, while maintaining the stability of the metal hardware, and could be a good solution to limit possible undesired interactions between proteins and column wall/frits. This prototype metal-free SEC column was systematically compared with a conventional stainless-steel SEC column hardware packed with the same stationary phase material. Four different mAb products, namely trastuzumab, palivizumab, bevacizumab and NISTmAb, and one antibody drug conjugate (ADC), trastuzumab emtansine, were selected as test samples. It appears that peak symmetry, separation of low molecular weight species (LMWS), and the recovery of high molecular weight species (HMWS) were significantly improved for the [...]

MURISIER, Amarande, et al . The importance of being metal-free: The critical choice of column hardware for size exclusion chromatography coupled to high resolution mass spectrometry.

Analytica chimica acta , 2021, vol. 1183, p. 338987

DOI : 10.1016/j.aca.2021.338987 PMID : 34627511

Available at:

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The importance of being metal-free: The critical choice of column hardware for size exclusion chromatography coupled to high resolution mass spectrometry

Amarande Murisier

^a^,^b

, Szabolcs Fekete

^a^,^b

, Davy Guillarme

^a^,^b

, Valentina D'Atri

^a^,^b^,^*

aInstitute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland

bSchool of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Prototype metal-free SEC column hardware are tested for mAb analysis in SEC-MS.

Prototypes are compared with standard stainless-steel SEC column hardware.

Prototypes show improved peak symmetry and size variants separation at LC level.

Performance of selected columns are evaluated in SEC-MS by using volatile mobile phase.

a r t i c l e i n f o

Article history:

Received 24 June 2021 Received in revised form 19 August 2021 Accepted 20 August 2021

Keywords:

Size-exclusion chromatography SEC-MS

Monoclonal antibody Metal-free stationary phase Bioinert stationary phase PEEK-Coated stationary phase Low adsorption stationary phase

a b s t r a c t

The goal of the study was to evaluate the possibilities offered by a new generation of metal-free SEC column to perform direct SEC-MS of protein biopharmaceuticals using ammonium acetate as the main mobile phase additive. The prototype metal-free SEC column hardware used in this work was a polyether ether ketone (PEEK) infused stainless steel tube including PEEK frits. This PEEK-lined column provides a fully bioinert and metal-freeﬂuidic path, while maintaining the stability of the metal hardware, and could be a good solution to limit possible undesired interactions between proteins and column wall/frits.

This prototype metal-free SEC column was systematically compared with a conventional stainless- steel SEC column hardware packed with the same stationary phase material. Four different mAb products, namely trastuzumab, palivizumab, bevacizumab and NISTmAb, and one antibody drug conjugate (ADC), trastuzumab emtansine, were selected as test samples.

It appears that peak symmetry, separation of low molecular weight species (LMWS), and the recovery of high molecular weight species (HMWS) were signiﬁcantly improved for the different biopharmaceutical products on the metal-free SEC column. It has also been demonstrated that the largest differences between standard and metal-free SEC columns were observed for the most basic mAbs (high pI), which conﬁrms that electrostatic interactions between the mAb and the metallic parts of the column (frits and inlet tube) could be responsible for the issues observed when performing SEC analysis with volatile mobile phase.

Finally, it was feasible to perform SEC-MS analysis for a wide range of biopharmaceutical products using volatile mobile phase. Our results also highlight that an inappropriate column could bias the

*Corresponding author. Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), School of Pharmaceutical Sciences, University of Geneva, CMU - Rue Michel Servet 1, 1211, Geneva 4, Switzerland.

E-mail address:[email protected](V. D'Atri).

Contents lists available atScienceDirect

Analytica Chimica Acta

j o u r n a l h o me p a g e : w w w . e l s e v i e r . c o m/ l o ca t e / a c a

https://doi.org/10.1016/j.aca.2021.338987

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quantiﬁcation of size variants when using MS-compatible mobile phases. Therefore, metal-free column, such as the PEEK-lined column, should be preferentially selected for SEC-MS analysis.

1. Introduction

Antibody-based drugs can undergo several enzymatic and chemical posttranslational modiﬁcations (PTMs) as well as degra- dations, resulting in variants that can be considered as critical quality attributes (CQAs) [1]. According to the ICH guidance Q8, CQAs are physical, chemical, biological or microbiological attributes that should be deﬁned, measured, and continuously monitored to ensure the desired quality of a drug product [2]. Among these CQAs, high molecular weight species (HMWS) resulting from therapeutic protein aggregation are particularly critical and require an accurate analytical characterization. These size variants can generate adverse immune reaction, alter the drug pharmacokinetics and reduce the potency of the desired biopharmaceutical product [3e5]. As stated in the monograph 129 of the United States Phar- macopeia (USP), size exclusion chromatography (SEC) is considered today as the reference method for the separation and quantitation of HMWS and low molecular weight species (LMWS) from the main isoform of therapeutic monoclonal antibodies (mAbs) [6].

In SEC, size variants are separated according to their hydrody- namic radii. The species arefiltrated through the stationary phase, comprised of spherical porous particles with a carefully controlled pore size [7]. Even though SEC is a non-retentive chromatographic mode, non-specific chemical interactions can occur between the biomolecules and the stationary phase, leading to protein adsorption, shifted elution time, peak asymmetry (tailing) and band broadening [8e10]. Additionally, mAbs are prone to irreversible adsorption on column material, resulting in a possible underesti- mation of the HMWS amount [7,11]. To reduce the unwanted hydrophobic interactions, organic solvent (isopropanol or acetonitrile) can be added to the mobile phase in small amount (5e15%), but there is a risk of protein denaturation. To eliminate, or at least reduce the electrostatic interactions, the salt concentration and ionic strength of the mobile phase need to be sufficiently high (i.e., phosphate buffer in combination with sodium chloride or potassium chloride), making SEC inherently incompatible with mass spectrometry (MS) [12e14].

Moreover, size variants characterization by SEC suffers from the difﬁcult identiﬁcation of the resolved chromatographic peaks [12].

For instance, species eluting before the main peak are considered as HMWS, but other species such as partially denatured monomers or oxidized monomer species may elute at the same time and could interfere with HMWS quantification [6,11,15]. The hyphenation of SEC with MS can overcome these drawbacks and additionally enable the detection of low-abundant species that were not detected by ultraviolet (UV) orfluorescence detectors [13,16]. For this purpose, different strategies have been developed in the last few years, based on two-dimensional liquid chromatography (2D- LC) coupled with MS [13,16e19]. Nevertheless, these procedures remain complex, time-consuming and require specially configured instruments [12,20]. Consequently, 2D-LC is not well adapted for routine application [13].

Hence, another trend that has recently emerged, consisting in coupling directly SEC with MS [17,20]. This hyphenation is possible when using alternative mobile phase, containing volatile salts

which are compatible with MS, such as ammonium acetate [17,19,20]. The direct SEC-MS coupling can be straightforward and ensure that proteins are analyzed in their native conformation [20].

Despite undeniable advantages, separation performance may be strongly compromised compared to classical mobile phases using high concentrations of non-volatile salts [12,13]. For instance, lower sensitivity, poor peak shape and limited aggregates recovery have been found in a previous study when analyzing a broad range of therapeutic proteins using direct SEC-MS with a state-of-the art SEC column, especially for basic proteins possessing isoelectric point (pI) higher than 7 [12]. Besides the mobile phase, the stationary phase was also highlighted as playing a signiﬁcant role in limiting unwanted interactions. Therefore, it has been concluded that further efforts should focus on developing metal-free SEC columns [12,20].

In the present study, we have tested a new generation of prototype metal-free SEC column, allowing the use of ammonium acetate buffer to perform a direct SEC-MS analysis [21]. The new metal-free column hardware was made of stainless-steel tube lined by polyether ether ketone (PEEK) and including PEEK frits.

The use of those columns in SEC-MS has never been reported elsewhere to our knowledge. The metal-free columns were systematically compared to their equivalent standard stainless-steel column hardware (packed with the same material), to evaluate their suitability for the measurements of HMWS. The peak tailing and aggregate ratio of several mAbs were assessed with MS- compatible mobile phases. Several mAbs and one antibody-drug conjugate (ADC) have been tested to illustrate the beneﬁts of metal-free column hardware.

2. Experimental

2.1. Chemicals and reagents

Type 1 water was provided by a Milli-Q puriﬁcation system from Millipore (Bedford, MA, USA). Ammonium acetate solution (Bio- Ultra, for molecular biology, ~5 M) was purchased from Sigma- Aldrich (Buchs, Switzerland).

2.2. Sample preparation

Therapeutic IgG mAbs, including, bevacizumab, palivizumab, trastuzumab and the ADC trastuzumab emtansine were obtained as European Union pharmaceutical-grade drug products from their respective manufacturers. The molecular weight (Da) of the mAbs and their isoelectric point are provided inTable S1. 7-year expired trastuzumab (Herceptin®, lot number B1605, expired on September 2014) and 11-year expired bevacizumab (Avastin®, lot number B2009, expired on May 2010) samples were available in our laboratory. NIST mAb reference material was purchased from the National Institute of Standards and Technology (Gaithersburg, MD, USA). Samples were diluted to 1 mg/mL in type 1 water for SEC experiments and to 10 mg/mL for SEC-MS experiments. Forced degradation under thermal stress of 10 mg/mL NIST mAb sample was perfomed by incubating the sample at 40C during 4 weeks.

A. Murisier, S. Fekete, D. Guillarme et al. Analytica Chimica Acta 1183 (2021) 338987

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2.3. SEC-UV experiments: instrumentation and experimental conditions

SEC analyses were performed on an ACQUITY UPLC™H-Class system (Waters, Milford, MA, USA), equipped with an auto-sampler including a 10m^Lﬂow-through-needle injector and a quaternary solvent delivery pump. Data were acquired using UV absorption at 280 nm. Both metal-free hardware, namely PEEK-lined columns, and standard columns of different pore sizes and particle sizes were used, as shown inTable 1.

The metal-free YMC-SEC MAB PEEK and the YMC-Pack Diol-200 PEEK columns (YMC Europe, Dinslaken, Germany) were prototypes not yet commercialized. They were operated at a ﬂow rate of 0.1 mL/min at room temperature. The MS compatible mobile phase was composed of 100 mM ammonium acetate in water (pH¼6.9).

These analytical conditions were applied to trastuzumab emtansine, trastuzumab, NIST mAb, bevacizumab and palivizumab, with the goal to compare the standard YMC-SEC MAB column and its metal-free version. Data acquisition and instrument control were performed by Empower Pro 3 software (Waters).

2.4. SEC-MS analyses: instrumentation and experimental conditions

The UHPLC system previously described was coupled to an ESI- Q-TOF mass spectrometer (Vion, Waters) to perform the SEC-MS analyses. The Vion was operated in the sensitivity mode and positive polarity to acquire continuum data in the range of 1000e16000m/z, with a scan time of 2 s. Capillary voltage was set at 3.0 kV, cone voltage at 150 V, and source offset at 80 V. Source temperature was set at 150C, desolvation temperature at 500C, and desolvation gasﬂow at 750 L/h. The system was calibrated by using a 200 pg/mL sodium iodide solution diluted in a mixture of water/isopropanol 50/50 (v/v) with 0.1% FA. UNIFI v1.9.4 was used for data acquisition, while protein mass spectra data treatment was performed with MassLynx software (Waters).

SEC-MS analysis were performed exclusively with the metal- free YMC-SEC MAB column kept at room temperature. The separation was carried out under isocratic conditions with 100 mM ammonium acetate as mobile phase, aflow-rate of 0.15 mL/min and injection volume of 10mL. A split of the LCflow was realized with a PEEK T-junction, so that theflow rate entering to the MS was equal to 0.05 mL/min.

3. Results and discussion

3.1. Impact of column characteristics on the separation of size variants

Two different metal-free SEC columns were compared for the analysis of NIST mAb size variants, as a representative example. The column dimensions were strictly identical (3004.6 mm), while only the particle size and pore sizes were different (2mm, 200 Å for the YMC-Pack Diol-200 and 3mm, 250 Å for the YMC-SEC MAB). In SEC, it is well known that particle size has an impact on

chromatographic efﬁciency, while pore size may alter the selectivity between size variants.

As illustrated inFig. 1, peaks were thinner on the YMC-Pack Diol- 200 column packed with 2mm particles, while the YMC-SEC MAB provides somewhat lower efﬁciency (N ¼ 14⁰850 and 11⁰807, respectively), but the performance remains acceptable in both cases. The impact of pore size was more signiﬁcant and the separations of HMWS and LMWS highlighted in the insets ofFig. 1were clearly different between the two columns. Indeed, the YMC-SEC MAB having nominal pore size of 250 Å offers a much higher selectivity for HMWS, with 2 peaks fully resolved from the main species. For this separation, the %HMWS was found to be 3.1%. On the other hand, the amount of HMWS observed on the YMC-Pack Diol-200 column was much lower (0.2% only), mostly due to its limited selectivity for the larger species. Expectedly, the reverse scenario was observed for LMWS, and the separation was much better on the column with smaller nominal pore size (YMC-Pack

Table 1

List of metal-free and standard SEC columns used in this work.

PEEK lined Column name Column dimension [mm] Particle size [mm] Pore size [Ǻ] Part number

Yes YMC-Pack Diol-200 metal-free 3004.6 mm 2 200 DL20S02-3046PTP^a

Yes YMC-SEC MAB metal-free 3004.6 mm 3 250 DLM25S03-3046PTP^a

No YMC-Pack Diol-200 3004.6 mm 2 200 DL20S02-3046PTH

No YMC-SEC MAB 3004.6 mm 3 250 DLM25S03-3046WT

aPrototype not yet marketed.

Fig. 1.Size variants analysis of the NIST mAb analyzed with two different SEC columns having different characteristics, namely YMC-SEC MAB PEEK (top panel) and YMC-Pack Diol-200 PEEK (bottom panel), and an MS-compatible mobile phase composed of 100 mM ammonium acetate. Experimental conditions are described in Section2.3.

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Diol-200). Here, the amount of LMWS was equal to 13.5% and two peaks were partially resolved from the main species.

Since HMWS species are more critical than LMWS in terms of toxicity and product efﬁcacy, a stronger focus was put on the separation of these species, and this is why the YMC-SEC MAB column was selected for the rest of the study [3]. However, it is important to keep in mind that the YMC-Pack Diol-200 can provide better separation of LMWS.

3.2. Standard vs. PEEK-lined columns in SEC

In one of our previous works, we have demonstrated that MS compatible mobile phase conditions were not suitable for the analysis of HMWS of any type of mAbs when using commercially available SEC columns. This was especially true when analyzing

basic mAbs having pIabove 7 [12]. At this time, we have highlighted the need to improve the stationary phase inertness, since this could be a viable solution to limit unwanted interactions leading to adsorption. Bioinert SEC columns are now developed by several providers, and therefore, our goal was to evaluate their performance in comparison with standard columns in a systematic way.

Fig. 2 shows the SEC analysis for various biopharmaceutical products, including four mAbs (i.e. trastuzumab, NIST mAb, bevacizumab and palivizumab) and one ADC (i.e. trastuzumab emtansine) which are known to be difﬁcult to analyze in SEC using ammonium acetate as mobile phase additive. These analyses were performed on the standard YMC-SEC MAB (right panel) and its metal-free counterpart (left panel).

When analyzing mAb products with standard stainless-steel SEC column (YMC-SEC MAB), signiﬁcant tailing was observed on

Fig. 2. Size variants analysis of four mAbs and one ADC by SEC, using a metal-free YMC-SEC MAB PEEK column (left panel) and its standard stainless-steel counterpart, by using an MS-compatible mobile phase composed of 100 mM ammonium acetate. Experimental conditions are described in Section2.3.

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the main peaks which may hamper the separation of LMWS eluted after the main species (USP tailing comprised between 1.4 and 2.2, as shown in Table 2). In addition, the peaks corresponding to HMWS and eluting before the main species were often not visible or only a very minor peak was observed (amounts comprised between 0 and 1% -Table 2), which may not correspond to the true amount of aggregates. This behavior can obviously be attributed to the presence of ammonium acetate in the mobile phase (relatively low ionic strength and weak buffer capacity at pH¼6.9) instead of the usual non-volatile SEC mobile phases including phosphate bufferþKCl at high concentration. Indeed, with this latter mobile phase composition, the differences in performance between standards and metal-free columns are not signiﬁcant as reported in Fig. S1.

The analyses were also performed on the metal-free SEC column and in this case, the peaks corresponding to the main species were more symmetrical (USP tailing comprised between 0.8 and 1.1 for mAbs, and 1.4 for ADC, as shown inTable 2) and it enabled to detect HMWS. Here, the amount of HMWS species was comprised between 0.3 and 4.5% (Table 2).

To better understand the differences observed between the two analytical SEC columns, we have explored the possible ionic and hydrophobic interactions that could take place during SEC analysis when the mobile phase is composed of ammonium acetate only.

First, it is important to consider that trastuzumab, NIST mAb and palivizumab have experimental pI(measured with icIEF) ranging from 9.1 to 9.3, while bevacizumab has a pIof 8.3 [22]. As shown in Fig. 2, it is clear that mAb products having the highest pIare the ones for which the largest differences were observed between the standard and the metal-free SEC column. On the other hand, the hydrophobicity (measured under HIC conditions) was the highest for bevacizumab, while the three other mAb products have a comparably low hydrophobicity [12]. Therefore, it is not possible to ﬁnd a direct correlation between hydrophobicity and adsorption of HMWS on the metal hardware. This conﬁrms that ionic interactions (and probably not hydrophobic interactions) between the mAb and the metallic parts of the column (frits and inlet tube) should take place and could be responsible for peak tailing of the main species and adsorption of HMWS on the standard YMC-SEC MAB column.

Obviously, this behavior is only present when the ionic strength and buffering capacity of the mobile phase is insufﬁcient (namely with 100 mM ammonium acetate as mobile phase constituent).

Those results ﬁnally highlight that an inappropriate column could bias the quantiﬁcation of HMWS when using MS-compatible mobile phases and that metal-free column should be preferentially selected for the analysis of size variants in SEC-MS.

3.3. Hyphenation to mass spectrometry: SEC-MS size variant identiﬁcation on selected samples and case studies

Based on the promising results reported in section3.2. with the metal-free SEC column, SEC-MS size variant analyses were performed on selected mAb products. First, to obtain the highest ESI- MS sensitivity while maintaining the best possible LC performance, a split of the LCﬂow was realized with a PEEK T-junction, so that theﬂow rate entering the ESI-MS was decreased from 0.15 mL/

min to 0.05 mL/min, as the best compromise between ESI-MS sensitivity, LC peak widths and total analysis time. The experiments were exclusively performed in the positive ESI ionization mode. Desolvation gas and source temperatures were carefully adjusted to obtain the proper solvent evaporation, while avoiding source activation and/or fragmentation. In addition, the influence of the injection volume on the MS sensitivity was also investigated (Fig. 3). Bevacizumab (10m^g/mL) was chosen as reference sample and injected at volumes of 2, 5, 10, 15, and 20mL, corresponding to sample mass load on column equal to 20, 50, 100, 150, and 200m^g, respectively. The variation of MS intensity as a function of injected sample was monitored by taking into account the MS spectra of the SEC main peak (specifically, the most abundant ion of the charge envelope, corresponding to the 27þcharge state). As reported in Fig. 3b, a column load of 100mg provided the highest MS sensitivity before a limited ionization capacity of ESI was reached. This setup was therefore used for the acquisition of the SEC-MS experiments of different mAbs. Specifically, the size variant analysis of NIST mAb, and the heat stressed NIST mAb; trastuzumab, and a 7-year expired trastuzumab sample; bevacizumab, and an 11-year expired bevacizumab sample were carried out (Fig. 4). In all cases, peak identification was performed by MS and the corresponding full list of elution times and mass assignments is reported inTable S2eS7.

Overall, a suitable separation of the main peak (denoted as M) from the HMWS and LMWS was obtained for these different samples.

Heat stressed NIST mAb (pink trace inFig. 4a) showed a higher percentage of LMWS (labelled as L1) alongside with a decrease of the main species compared to the non-stressed sample. Similarly, Table 2

Recovery of LMWS/HMWS and USP tailing obtained by SEC for four mAbs and one ADC using a metal-free YMC-SEC MAB PEEK column (left panel) and its non-metal-free counterpart, a YMC-SEC MAB (right panel), with an MS-compatible mobile phase composed of 100 mM of ammonium acetate. Experimental conditions described in Sec- tion2.3.

YMC SEC-MAB PEEK YMC SEC-MAB

NIST mAb Amount of HMWS (%) 3.1 0

Amount of the main peak (%) 96.9 100

Amount of LMWS (%) 0.0 0

USP Tailing 1.1 2.2

Bevacizumab Amount of HMWS (%) 3.1 1.0

Amount of the main peak (%) 94.3 98.5

Amount of LMWS (%) 2.5 0.5

USP Tailing 0.9 1.7

Palivizumab Amount of HMWS (%) 4.5 0.3

Amount of the main peak (%) 94.1 90.5

Amount of LMWS (%) 1.4 9.2

USP Tailing 1.0 1.4

Trastuzumab Amount of HMWS (%) 0.3 0

USP Tailing 0.8 1.5

Trastuzumab emtansine Amount of HMWS (%) 0.7 0

USP Tailing 1.4 1.9

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the 7-year expired trastuzumab sample (red trace inFig. 4b) presented a signiﬁcant increase amount of LMWS (denoted as L1).

Finally, the most interesting size variant profile was obtained for bevacizumab (grey trace inFig. 4c). Indeed, this sample presented a HMWS that was identified as a dimer, and two LMWS identified as the Fc-Fab (labelled as L1) and Fab (labelled as L3) fragments, respectively. Intriguingly, the 11-year expired bevacizumab sample (blue trace in Fig. 4c), showed an increased amount of the two LMWS, with an additional size variant corresponding to the Fc fragment (labelled as L2). Despite the very good LC separation of all the size variants, it should be noted that several glycoforms and proteoforms are hidden below each peak. As shown in Fig. 5for bevacizumab size variants analysis, the peak corresponding to the monomeric species (red trace inFig. 5c) was indeed characterized by the co-elution of three different glycoforms, namely G0F/G0F, G0F/G1F, and G1F/G1F, all containing the clipping of the C-terminal

Lys (labelled as 0K). The same glycoforms and Lys clipping were identified for the Fc-Fab fragment (blue trace inFig. 5c) that additionally presented different proteoforms generated by different cleavage sites. Similarly, different proteoforms were detected for the Fab fragment (green trace inFig. 5c). Indeed, as highlighted by the cartoon reported inFig. 5, the heavy chain upper hinge region generally subjected to cleavage consists of the amino acid sequence SCDKTHTCP, where thefirst Cys residue is involved in the inter- chain disulphide bond linking together the heavy and the light chains, while the second Cys links together the two heavy chains [23]. This fragmentation site can give rise to different Fab and Fc- Fab fragments that only differ in a few amino acids, depending on where the cleavage is occurring, and is obviously responsible for the complex mass profiles reported inFig. 5c. However, it should be noted that this fragmentation pathway is quite recurring and it was indeed identified for the LMWS of all the samples analyzed in this study, as reported inTable S2eS7.

Fig. 3.SEC-MS method optimization: inﬂuence of injection volume. (a) Total ion chromatograms (TIC) of bevacizumab sample by using injection volumes of 2, 5, 10, 15, and 20mL, corresponding to different sample mass loads on column (namely, 20, 50, 100, 150, and 200mg). (b) Variation of MS intensity of SEC-MS as a function of injected bevacizumab sample. Reported values refer to the MS intensity of the main peak (speciﬁcally, the 27þcharge state of its mass spectra). Experimental conditions are described in Section2.4.

Fig. 4.Size variants analysis of different mAbs by SEC-MS analysis. Total ion chromatograms (TICs) and zoom on minor peaks of (a) NIST mAb (green line) and stressed NIST mAb (pink line), (b) trastuzumab (black line) and a 7-years expired trastuzumab sample (red line), and (c) bevacizumab (grey line) and an 11-years expired bevacizumab sample (blue line). TIC peaks identiﬁed as main (M), high molecular weight species (HMWS), or low molecular weight species (LMWS, labelled as L1-L3) variants.

For sake of simplicity, mAb cartoons were added on the top of selected peaks.

Experimental conditions are described in Section2.4. (For interpretation of the references to color in thisﬁgure legend, the reader is referred to the Web version of this article.)

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4. Conclusions

In this work, newly developed metal-free SEC columns were evaluated for the analytical characterization of protein biopharmaceuticals, including mAbs and ADCs. In this column, PEEK was used as liner on the inner wall of the column tubing and frits were also made of PEEK. Such a column provides metal-freefluidic paths, while maintaining the mechanical stability of the metal hardware. The metal-free column was systematically compared to its stainless-steel counterpart for the size variant analysis of various mAbs and ADC samples. As expected, significant differences were observed in terms of HMWS recovery and monomer peak shape (severe tailing). This confirms that chemical/adsorptive interactions between sample and chromatographic metallic surface can be discarded when using a metal-free column hardware. In addition, we have also demonstrated that the differences observed between the standard and metal-free columns were particularly substantial for the most basic mAb products (i.e. NISTmab, trastuzumab and palivizumab). This confirms that electrostatic interactions may be responsible for adsorption phenomenon on the metallic parts of the column (inlet tube and frits), when using a volatile mobile phase having limited buffering capacity (100 mM ammonium acetate at pH¼6.9).

Finally, the metal-free column was successfully applied to perform direct SEC-MS experiments of regular and stressed/

expired mAb products. Differences in terms of size variant proﬁle were clearly found between the two types of samples, and the different peaks observed were easily identiﬁed thanks to high resolution MS.

CRediT authorship contribution statement

Amarande Murisier:Investigation, Data curation, Visualization, Writingeoriginal draft.Szabolcs Fekete:Conceptualization, Su- pervision, Writinge review&editing.Davy Guillarme:Concep- tualization, Supervision, Funding acquisition, Writing e original draft, Writing e review & editing. Valentina D'Atri: Project administration, Investigation, Data curation, Visualization, Writing eoriginal draft, Writingereview&editing.

Declaration of competing interest

The authors declare that they have no known competing ﬁnancial interests or personal relationships that could have appeared to inﬂuence the work reported in this paper.

Acknowledgements

The authors wish to thank Daniel Eber (YMC Europe GmbH, Dinslaken, Germany) for providing the columns used in this work;

Henri Kornmann and Kudzai Mutenda (Ferring Pharmaceuticals, Saint-Prex, Switzerland) for theﬁnancial support of this work; and Jean-Luc Veuthey from the University of Geneva for his fruitful comments and discussions.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2021.338987.

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