Determination of ultra-trace metal-protein interactions in co-formulated monoclonal antibody drug product by SEC-ICP-MS

ABSTRACT

Transition metals can be introduced in therapeutic protein drugs at various steps of the manufacturing process (e.g. manufacturing raw materials, formulation, storage), and can cause a variety of modifications on the protein. These modifications can potentially influence the efficacy, safety, and stability of the therapeutic protein, especially if critical quality attributes (CQAs) are affected. Therefore, it is meaningful to understand the interactions between proteins and metals that can occur during the manufacturing process, formulation, and storage of biotherapeutics. Here, we describe a novel strategy to differentiate between ultra-trace levels of transition metals (cobalt, chromium, copper, iron, and nickel) interacting with therapeutic proteins and free metal in solution in the drug formulation using size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC-ICP-MS). Two monoclonal antibodies (mAbs) were coformulated and stored up to nine days in a scaled down model to mimic metal exposure from manufacturing tanks. The samples containing the mAbs were first analyzed by ICP-MS for bulk metal analysis, then studied using SEC-ICP-MS to measure the extent of metal-protein interactions. The SEC separation was used to differentiate metal associated with the mAbs from free metal in solution. Relative quantitation of metal-protein interaction was then calculated using the relative peak areas of protein-associated metal to free metal in solution and weighting it to the total metal concentration in the mixture as measured by bulk metal analysis by ICP-MS. The SEC-ICP-MS method offers an informative means of measuring metal-protein interactions during drug development.

KEYWORDS:

• Co-formulation
• drug formulation
• mAbs
• SEC-ICP-MS
• ultra-trace metal analysis

Introduction

Transition metals found in biological systems are essential to key functions, including maintenance of protein structural integrity and catalytic enzyme activity.^1,2 However, the presence of transition metals (e.g. Fe, Cu, Cr, Co and Ni) in therapeutic protein drug products can also serve as catalysts for a variety of interactions with proteins, including the generation of reactive oxygen species (ROS) that cause oxidation and result in modifications to proteins that can affect their stability and function.³ The impact of metals on protein oxidation is of primary importance for therapeutic proteins such as monoclonal antibodies (mAbs)^4–8 because these type of molecules are an important class of biologic used as therapeutic agents in areas such as oncology, anti-inflammatory, and autoimmune disease.^9,10 Therapeutic mAbs are particularly susceptible to oxidative stress from the presence of trace metals introduced at various stages of manufacturing, and various sources, such as raw materials contamination, glass vials, rubber stoppers, and stainless steel, can introduce the trace metals.^11,12 Studies have shown that the protein concentration is a statistically significant factor impacting the metal leachability when in the presence of stainless steel (SS) because the protein can act as a complexing agent for metal ions, which suppresses the formation of a protective oxide layer on the surface of the SS.^13,14 Brown and Merritt reported that the presence of protein produced corrosion on the outer layer of SS.¹⁵

These transition metals can achieve multiple oxidation states, which increases the potential to generate ROS by facilitating the reduction of molecular oxygen, leading to radical generation and oxidative damage of the mAbs.^16,17 Copper and iron in particular can cause production of free hydroxyl radicals by Fenton reactions. Indeed, Cu²⁺ and Fe³⁺ reduce sulfur-containing amino acids like cysteine and methionine, forming disulfide bonds and releasing Cu¹⁺ and Fe²⁺ ions, which can then react with hydrogen peroxide from thiol compounds used as anti-oxidants and release hydroxyl radicals.^18,19 This oxidative damage has the potential to affect the quality of the drug products if it affects efficacy and critical quality attributes (CQAs) of the molecule. Many studies have highlighted the modification of amino acids by trace metals such as Fe²⁺.^20–24 For example, Ouellette et al. revealed that fragmentation near the hinge region of four IgG1 molecules was catalyzed by the synergistic reaction of Fe²⁺ and histidine.²⁵ A similar study was performed by Glover and coworkers with copper, where they demonstrated the damaging interaction between copper and histidine residues of an IgG1 molecule, which also led to fragmentation at the hinge region.²⁶ The Food and Drug Administration requires monitoring and control of trace metal elements in drug formulations, but Pistacchio et al. highlighted that keeping the levels of both copper and iron below the allowed limits does not necessarily guarantee stable physicochemical properties and can lead to oxidation.²⁷

The detection of ultra-trace transition elements in drug product formulation and their potential interaction with the proteins is thus crucial to understanding modifications of biotherapeutic drugs. Conventional inductively coupled plasma-based methods such as atomic emission spectrometry or mass spectrometry (ICP-AES and ICP-MS, respectively) allow detection of trace metals in the drug product, but can only provide information about total metal content in solution. Methods that provide information about metal speciation, which can be used to understand the interactions between metals and proteins, are thus needed for biologics development.²⁸ Other methods such as native MS are well-suited to measure specific metal-protein binding,^29–32 but are generally not suitable to measure the total amount of free metal that may interact with a protein in solution. The coupling of high-performance liquid chromatography (HPLC) and ICP-MS has become the preferred tool to study such interactions.^33–35 This is principally due to the variety of available chromatographic separation methods, and the sensitivity of element-specific detection offered by an ICP-MS. The combination of HPLC and ICP-MS allows both identification and quantitation of specific metals within each chromatographically resolved species.

Many studies have used HPLC-ICP-MS for the quantification of proteins,^36,37 and a number of these studies have used SEC-ICP MS with single or multiple element detection in biological matrices.^38–41 For example, Richarz and Brätter led a study where SEC-ICP MS was used to measure different elemental profiles in cytosols of cells from Alzheimer patients’ brains by analyzing copper, zinc, cadmium, lead, and silver. In their study, they compared the metal levels in the cytosols of brains with Alzheimer’s disease and control brains and discovered that there was an increased level of oxidized metallothioneins in Alzheimer’s disease brains.^42,43 Gailer and coworkers demonstrated the coupling of HPLC and SEC-ICP-AES and performed the simultaneous analysis of copper, iron and zinc species in rabbit plasma.³⁵ The relatively high limit of detection of the ICP-based instrument used in that study did not hinder the analysis, as the metal concentration of the species found in the plasma samples ranged from 0.2 mg/L to 3.0 mg/L. Overall, their method allowed the detection of 12 metalloproteins and metallopeptides in less than 25 min. These studies highlight the utility of SEC-ICP-MS to measure metal-protein interactions in complex matrices. However, the metal concentrations found in a formulated drug product will be significantly lower than those in blood, plasma or urine, which means that previously reported methods are not sensitive enough to detect metals in formulated drug product.⁴⁴ Therefore, there is a need for an accurate and highly sensitive method that can identify metal-protein interaction at ultra-trace levels.

Herein, we describe an SEC-ICP-MS method capable of measuring metal-protein interactions present in a therapeutic drug product consisting of two co-formulated mAbs at ultra-trace levels. We first use ferritin and Cu,Zn-superoxide dismutase, two metalloproteins containing transition metals, to develop the method and provide proof of concept. We then analyzed co-formulated mAbs stressed using stainless steel coupons to simulate the level of metal that may be introduced through drug substance manufacturing. Moreover, we calculate the relative quantitation of metal-protein interaction and free-metal, using a relative peak area quantification approach combined with total metal determination. Our technique led to an improved understanding of metal-protein interactions in biologic formulations, and more importantly serves as a tool to better understand the role of metal-protein interactions in metal-induced protein degradation often associated with biologics manufacturing.

Results

Demonstration of the SEC-ICP-MS capability using metalloproteins

The first step of the study was to evaluate and optimize the separation of protein-associated metals and free metals species. Ferritin and Cu,Zn-superoxide dismutase (SOD) were chosen as appropriate metalloproteins for method development because of their high level of associated transition metals and diverse molecular weights. Both standards were used to optimize column choice and flow rates eventually leading to the parameters outlined in the LC-MS Method section.

Figure 1a illustrates the separation of two distinct peaks for the ferritin, eluting between 2.9 and 3.4 min in the UV trace, which was achieved using the final chromatographic conditions. In the ICP-MS chromatogram, the first peak, which appears at 2.9 min, corresponds to the aggregated (high molecular weight) species while the second and main peak eluting at 3.4 min corresponds to ferritin monomer, and matches the profile and intensities observed in the UV trace.⁴⁵ The retention time of the main ferritin peak, which corresponds to a molecular mass of 480 kDa, is aligned with the observations in the literature.^46,47 A calibration curve using ferritin at iron concentrations varying between 5.0 and 100 µg/L was also generated and demonstrated good linearity within that range (Figure S2).

Figure 1. Chromatograms by SEC-UV (280 nm) and SEC-ICP-MS detection of a) equine ferritin and b) bovine Cu, Zn-SOD (UV trace off axis for clarity purposes).

Two chromatograms of metalloproteins are shown to demonstrate proof-of-concept. The chromatogram on the left (A) shows agreement between the retention time of the UV and SEC-ICP-MS traces of ferritin. The chromatogram on the right (B) shows agreement in retention time of the UV and SEC-ICP-MS traces of Cu,Zn-SOD.

In addition to ferritin, Cu,Zn-SOD was analyzed to evaluate the chromatographic conditions (Figure 1b). By comparing the profiles of both the UV and SEC-ICP-MS traces, the major peak at 4.2 min exhibits both UV absorbance, and Cu and Zn signal. However, the UV trace also reveals that the standard chosen is not fully pure and contains other absorbing species at a retention time of 4.4 min without association of Cu and Zn ions. The peak areas of each resolved peak were evaluated, and the corresponding Cu,Zn-containing species accounted for 55% of the total area. This implies that the protein standard contains species other than the metalloprotein that account for about 45%.

Study of metal-protein interaction in co-formulated mAbs

Total content of metals in monoclonal antibodies SS study samples

Transition metals can be introduced in the manufacturing process through various means, such as raw material contamination, and direct contact with unit operations. The most commonly used grade of stainless steel used in biopharmaceutical applications is 316 L, which is an alloy containing mainly chromium, iron, and nickel, with minor amounts of copper.^11,48 The stainless steel contact through unit operations is a major source of the metal contamination in biologics drug products, as this contact takes place in various tanks and stainless steel piping.¹³ During manufacturing and storage, biologics formulations are in contact with stainless steel at different fill volume per unit contact surface areas. In this study, a smaller volume per unit contact surface (2.0 mL/cm² was chosen to represent a high level of interaction between the drug product and the SS coupons and incubation occurred for up to 9 days. In large-scale production, tanks generally have higher volume per unit surface, of the order of 10 mL/cm² or more. Bulk metal analysis of the four stainless steel-stressed co-formulated mAb samples was performed in triplicate by ICP-MS/MS following the acid digestion protocol. The total concentrations of metals of interest are reported in Table 1.

Table 1. Total metal content in mAb stress study samples (n = 3, 95% confidence interval).

Sample	Concentration (µg/L)
	Co	Cr	Cu	Fe	Ni
t=0	6.3 ± 0.2	1.9 ± 0.1	0.8 ± 0.1	8.2 ± 0.2	2.5 ± 0.1
5 days	6.5 ± 0.2	5.9 ± 0.2	16 ± 0.2	55 ± 1.6	19 ± 0.3
7 days	6.5 ± 0.2	6.4 ± 0.2	19 ± 0.2	88 ± 2.4	16 ± 0.3
9 days	6.4 ± 0.1	7.1 ± 0.2	18 ± 0.1	87 ± 2.3	16 ± 0.3

Compared to the t=0 sample, the samples with the SS coupons showed higher levels of chromium, iron, and nickel, which is consistent with the composition of SS 316 L, and the specified metal leached over the course of the experiment. Interestingly, the cobalt concentration did not change, probably because this element is not present in high concentration in the SS grade used in the study.

Metal speciation in mAb samples by SEC-ICP-MS

The measurement of metal-protein interaction was conducted for the stressed co-formulated mAb samples. The samples were first diluted to a final protein concentration of 1 mg/mL before injecting 5 µL into the SEC-ICP-MS system. These conditions allowed partial separation of the co-formulation mAbs, as displayed in the UV spectra obtained at 280 nm (Figure 2). Although the primary separation mechanism of SEC is based on hydrodynamic radius, the partial separation of the co-formulated mAbs suggests that secondary interactions such as differences in hydrophobicity of the two molecules also affect the separation, as described in the work of Yan et al.⁴⁹ The separation of the two mAbs was confirmed by measuring the molecular weight of each peak by native SEC-MS (Figure S1 and Table S3).

Figure 2. UV chromatograms of a) 10 µg loading that allows for co-formulation separation and b) 400 µg loading that allows for metal-protein binding affinity analysis.

Two UV chromatograms at 280 nm showing the effect of sample loading on the co-formulation separation. A) Two peaks corresponding to each mAb in the are distinguished with a sample loading of 5 µg. B) A single peak saturating the detector signal is visible with a 400 µg injection load.

While loading the column with a total of 5 µg of protein allowed UV detection of the co-formulated mAbs (Figure 2a), it did not permit the detection of metal-protein interaction by ICP-MS, as only background noise was detected due to the low level of metal present in the samples. Therefore, the injection amount was increased 80-fold to 400 µg of protein injected on column. This increase in the protein loading on the column resulted in peak broadening and saturation of the detector (Figure 2b). Moreover, as we increased the loading, a small peak is detected at 2.9 min, indicating the detection of minor aggregate species.

The optimized loading of SS-stressed mAb samples helped facilitate the detection of protein interaction with chromium, cobalt, nickel, and iron, as depicted in Figure 3. The SEC-ICP-MS chromatograms obtained for chromium, cobalt, and nickel (Figure 3a–c) consisted of a single peak between 3.19 and 3.83 min, which correlated with the protein retention time detected by UV. Later eluting peaks were observed between 5 and 7 minutes, which indicates free metal in solution that is eluting in the permeation limit of the column. In the case of the Fe chromatogram (Figure 3d), two distinct peaks in the time-range of protein elution indicates association of Fe with the protein, including a peak that likely represents protein aggregate at 2.85 min and the main peak at 3.45 min. Similar to chromium, cobalt and nickel, late-eluting species containing Fe were observed between 5.15 and 7.40 min, indicating free iron or iron associated with smaller molecules. Further investigation is under way to identify the free iron species leading to the presence of two peaks in the free iron region. The analysis of the formulation buffer without mAb confirmed the retention time of the free metal species in the formulation (Figure S3).

Figure 3. SEC-ICP-MS chromatogram of various time point of the SS samples analyzed for a) chromium, b) cobalt, c) nickel, and d) iron.

Four panels are displayed plotting the metal intensity with retention time. The SEC-ICP-MS trace of A) chromium B) cobalt C) nickel D) iron. The earlier retention of the metal coincides with that of the mAbs suggesting the presence of metal-protein interaction. Nickel displays the least interaction with the co-formulated mAbs while iron displays the most interaction with the co-formulated mAbs.

The SEC-ICP-MS chromatograms of the various time points of the SS study indicate a general increase in the peak area over time. For chromium, cobalt and nickel, there was an increase in the peak area only in the late-eluting, free metal species region, while the opposite was observed for iron. Indeed, the free iron species peak areas (RT = 5.2–7.4 min) were nearly unchanged, while the peaks indicating mAb-iron interaction increased (RT = 2.8–4.0 min), suggesting that the mAb interaction with iron selectively accumulates over time, but not for the other three metals (Figure 3).

Identification of copper-histidine interactions

During our analysis, we observed an artificially high peak area at t = 0 sample in the copper chromatograms in the free copper region (~5.5 min), as depicted in Figure 4a. Further study suggested that the presence of histidine in the formulation buffer resulted in copper ions leaching from the HPLC system. Indeed, among the amino acids, L-histidine is one of the strongest copper-chelating ligands, as demonstrated by Deschamps and coworkers.⁵⁰ Prior to the addition of a wash step, the peak area of copper did not correlate with the total copper content obtained by bulk ICP-MS/MS analysis (Table 1), with the highest peak area being for t = 0 sample (Figure 4a). As samples were injected, the peak area decreased, indicating that less copper was stripped from the HPLC components as the system was continuously flushed with mobile phase. In an attempt to counteract this phenomenon, we added an L-histidine washing step before and between runs to remove any contribution of copper leaching from the instrument. The implementation of the histidine wash step solved this issue and generated results that agreed with the trends observed by bulk ICP-MS/MS analysis (Table 1), as illustrated in Figure 4b. It was then possible to observe that the copper-protein interaction peak area increased from t = 0 to 5 days in the mAb eluting range and further stabilized after 5 days. Similar to chromium, cobalt and nickel, a peak increase for copper was detected in the free metal species region at the later eluting range.

Figure 4. SEC-ICP-MS chromatogram of copper at various time point of the SS incubated samples a) without the L-histidine washing step showing artificially high peak areas in the free metal species regions and b) with the L-histidine washing step resolving the artificially high peak areas.

SEC-ICP-MS chromatograms of copper are displayed. On the chromatogram on the left, A) the copper peak in the free metal species region is artificially high for t=0 sample. An insert of the copper-protein interaction shows the low intensity interaction of the metal with the co-formulated mAbs. B) The copper peak in the free metal species region is now aligned with the ICP-MS/MS bulk analysis. Insert, the retention time of copper coincides with that of the mAbs suggesting presence of copper-protein interactions.

Calculation of peak area distribution of SS stressed mAbs

Relative semi-quantitation of the metal concentration for each species detected by SEC-ICP-MS was calculated using a relative peak area quantification approach adapted from del Castillo-Busto and coworkers,⁵¹ which does not require standards. Briefly, the sum of the peak areas from the metal species in the co-formulation (Table S4) was set equal to the total concentration of metals (μg/L) obtained by bulk ICP-MS/MS analysis (Table 2). Using this approach, we assume that the mass balance of the bulk analysis is preserved when performing the SEC-ICP-MS analysis i.e., no metal is lost from the sample during the analysis, or the sample is not contaminated when introduced in the system. The concentrations of each metal species (M) and their relative proportions in the SS study samples were calculated following equation 1(1) $Concentratio{n}_{metalM}=\frac{Peak\hspace{0.17em}Are{a}_{metal\hspace{0.17em}M}\times Total\hspace{0.17em}meta{l}_{}ICP}{\sum Peak\hspace{0.17em}Area{s}_{}metal\hspace{0.17em}M}$(1) :

Table 2. Relative semi-quantitation of metal associated to mAbs in coformulated SS study by SEC-ICP-MS using the combination of metal-protein peak area distribution and total metal results by ICP-MS/MS.

Sample	Concentration (µg/L)
	Cr		Co		Cu		Fe		Ni
	Assoc.	Free	Assoc.	Free	Assoc.	Free	Assoc.	Free	Assoc.	Free
t=0	0.7 ± 0.04	1.2 ± 0.1	0.5 ± 0.02	5.8 ± 0.3	0.0 ± 0.01	0.8 ± 0.1	4.0 ±0.2	4.2 ± 0.2	0.1 ± 0.01	2.4 ± 0.1
5 days	1.5 ± 0.1	4.4 ± 0.2	0.5 ± 0.02	6.0 ± 0.2	0.6 ± 0.01	15 ± 0.6	30 ± 1.5	25 ± 1.4	0.3 ± 0.01	18 ±0.6
7 days	1.6 ± 0.1	4.8 ± 0.3	0.5 ± 0.03	5.9 ± 0.3	0.7 ± 0.02	18 ± 0.4	60 ±2.4	28 ± 1.7	0.3 ± 0.01	16 ± 0.7
9 days	1.4 ± 0.1	5.6 ± 0.2	0.5 ± 0.02	5.9 ±0.2	0.6 ± 0.02	17 ± 0.7	55 ± 2.2	32 ± 1.4	0.3 ± 0.02	16 ± 0.8

(1) $Concentratio{n}_{metalM}=\frac{Peak\hspace{0.17em}Are{a}_{metal\hspace{0.17em}M}\times Total\hspace{0.17em}meta{l}_{}ICP}{\sum Peak\hspace{0.17em}Area{s}_{}metal\hspace{0.17em}M}$(1)

The results obtained by the peak area distribution quantification approach for the SS study samples are summarized in Table 2. The results demonstrate that a small proportion of chromium, cobalt, copper, and nickel is associated with the mAbs, but that the majority of these metal-species found in the samples are of small molecular weight and free in solution. Iron exhibits the opposite behavior where the concentration of iron-associated species increased with time of SS exposure, whereas the concentration of low molecular weight iron species stayed nearly constant.

In order to confirm that the increase in the concentration of iron associated with the proteins was not specific to the samples tested, we analyzed two additional co-formulated mAbs that followed the same SS study protocol. The identical phenomenon was observed again as depicted in Figure 5, indicating the tendency of iron to associate with proteins rather than accumulate in solution.

Figure 5. Iron chromatograms depicting the increase of Fe-associated species over time in co-formulation a and co-formulation b.

SEC-ICP-MS chromatograms depicting the increase of Fe-associated species over time in co-formulation A (left side) and co-formulation B (right side). The free iron species peak does not increase over time.

Discussion

The analytical task of determining the interactions of metal and proteins, and their impact on formulated drug products, can be simplified using the right tools. To this end, we developed the novel ultra-trace multi-element SEC-ICP-MS method to gain insight on metal-protein interactions that could lead to oxidation and affect the CQAs of the drug. The combination of SEC and ICP-MS permits the separation of metal-containing species in formulated drug products according to their size and provides valuable data that cannot be obtained by conventional ICP-MS methods or other assays. Indeed, determination of the total concentration of metals in formulated drug products does not provide enough information about the distribution of ultra-trace levels of metals among proteins and small molecular weight species.

This novel SEC-ICP-MS method allows fast and efficient separation of high and low molecular weight species in co-formulated mAbs. In co-formulated samples, the injection of 5 µg of mAbs using the chromatographic conditions we developed enabled partial separation of the two mAbs. In order to detect ultra-trace levels of protein-metal interactions, the loading was increased to 400 µg, which allowed detection of metal species with the same retention time as the mAbs in the co-formulated drug product, strongly suggesting interactions between Fe and the mAbs in solution. These interactions were also observed in two other co-formulated mAbs, indicating that the phenomenon is related to the metal rather than the mAbs or the formulations. However, further investigation is needed to better understand the behavior of free iron in the sample formulation. Sensitivity to L-histidine contained in the formulation of the drug product was observed, artificially elevating the free copper peak intensity, which was remedied with a histidine washing step. Although the mechanism remains unclear, our observations suggest that protein-metal interactions may occur upon exposure of protein to stainless steel during the manufacturing process and should be closely monitored.

We believe that the technique developed here will be an invaluable tool to monitor mAb manufacturing and to support studies designed to understand metal-induced protein aggregation and modification in biologic products. Moreover, it can facilitate deeper understanding of the impact of metal exposure during drug development and manufacturing.

Materials and methods

Materials

Purified water produced using a Milli-Q system (Millipore, Bedford, MA) was used to prepare all solutions. All chemicals and reagents were trace-metal grade and used without further purification. Ammonium acetate (99.999% trace metal basis), L-histidine (BioUltra grade), type 1 equine ferritin (Catalog no. F4503-100 MG) and bovine Cu,Zn-superoxide dismutase (Cu,Zn-SOD, catalog no. S7571-15KU) were all purchased from Sigma Aldrich (St. Louis, MO). The HPLC mobile phases consisted of 200 mM ammonium acetate at a pH of 6.5. The washing mobile phase consisted of 10 µM L-histidine buffer in water.

A multi-element standard at a nominal concentration of 10 mg/L (High Purity Standard, North Charleston, USA) was used to prepare calibration standards in mobile phase and used to characterize free species. Both type 1 equine ferritin and bovine Cu,Zn-SOD were prepared by diluting the appropriate amount of standard solution in ammonium acetate buffer to achieve a final protein concentration of 140 µg/mL and used as control material for protein-associated metal species.

Equipment

An Agilent 8900 ICP-MS/MS (Santa Monica, CA) equipped with a Micro Mist concentric nebulizer and a Scott double-pass spray chamber was used for the study. The chromatographic system for SEC-ICP-MS experiments was an Agilent 1290 HPLC (Santa Monica, CA) with a variable loop (5–20 µL) and a diode array detector (DAD). The HPLC system was coupled to the ICP-MS/MS through an interface consisting of a 45 cm PEEK capillary tubing to connect the column outlet to the nebulizer inlet. The SEC column was an Acquity UPLC Protein BEH SEC column (200 Å, 1.7 µm, 4.6 × 150 mm, 1.7 µm, Waters, Millford, MA) Native MS experiments were carried out on a Orbitrap Eclipse mass spectrometer coupled to a Vanquish Flex UPLC with a variable wavelength detector (Thermo Scientific) using the same SEC column and mobile phase as SEC-ICP-MS experiments. A “resistor tube” was used between the ESI probe and ground to lower the electrospray current, similar to that described previously.⁵²

LC-MS methods

Separation for SEC-ICP MS experiments of ⁵²Cr, ⁵⁶Fe, ⁵⁹Co and ⁶⁰Ni was performed over 8 min using an isocratic flow of 200 mM ammonium acetate (pH unadjusted) at 350 µL/min. The column was first conditioned by passing at least 10 mL of mobile phase through it before injection of the standards and samples. Isocratic elution was used to separate both free and associated metal species. For the analysis of ⁶⁵Cu, the separation was performed as follows: an initial isocratic flow of 200 mM ammonium acetate for 8 min at 0.35 mL/min followed by a wash step using 10 mM of L-histidine at a flow rate of 0.5 mL/min, and then equilibration with 200 mM ammonium acetate for 7 min back to starting conditions to prepare the column for the next injection.

Injection amounts were 400 µg for mAb samples and 10 µg for both ferritin, Cu,Zn-SOD and free metal standard. The ICP-MS/MS instrument settings (nebulizer gas flow, RF power and lens voltage) were checked and tuned daily using Agilent tuning solution. The isotopes monitored for mAb-metal interaction experiments included ⁵²Cr, ⁵⁶Fe, ⁵⁹Co, ⁶⁰Ni and ⁶⁵Cu. The argon-based and other polyatomic interferences⁵³ were successfully removed by operating the ICP-MS in MS/MS mode and with the introduction of 3 mL/min of helium gas in the collision cell.

Peak areas of the separated metal species were processed using Agilent MassHunter software. The operating conditions for SEC-ICP-MS are summarized in Table S1. Native SEC-MS experiments were performed with the same separation conditions with an additional switching valve used to divert nonvolatile salts to waste after protein elution. The MS parameters for native MS experiments can be found in Table S2.

Monoclonal antibody stainless steel study

The mAb co-formulation was prepared by mixing equal portion of mAb A, an IgG1, and mAb B, an IgG4, to achieve a final protein concentration of 20 mg/mL. The co-formulation was divided and dispensed into four 30 mL sterile polyethylene terephthalate glycol (PETG) media bottles separately. Thirty 316 L grade stainless steel (SS) coupons (Stollen Machine & Tool, Kenilworth, NJ) of 2 cm² were divided equally and added to three of the sample bottles, while the last one acted as negative control. All three bottles containing the SS coupons were staged on stability at 25°C over time points of 5, 7, and 9 days. Once a stability time point was reached, the bottle was removed from the 25°C chamber and the sample was transferred (without coupons) to a sterile PETG media bottle. The process was repeated for each time point. All samples were stored at 5°C until prior to SEC-ICP-MS testing.

Acid digestion of SS study samples for ICP-MS/MS analysis

For sample digestion, a gentle acidic digestion protocol using a hot block was developed. In short, 250 μL of sample was mixed with 250 μL of a digestion solution containing equal parts of nitric acid and hydrogen peroxide in a metal-free polystyrene tube. This solution is then gently heated to a maximum of 70°C for up to 1 h to break down proteins that would interfere with the measurement. After compensation for volume loss, the clear solution was diluted with Milli-Q water to a dilution factor of 10, which allows for the determination of ultra-trace elements in mAb samples by ICP-MS/MS.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2199466

Additional information

Funding

This work was funded by Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA.