1. Introduction
Therapeutic monoclonal antibodies (mAbs) are a major class of biopharmaceuticals which have seen rapid growth in recent decades. They have an immunoglobulin (Ig) structure that is capable of specific binding to unique epitopes present in their target. As a consequence, mAbs have notably improved the treatment results for a wide variety of human-specific diseases such as cancer, autoimmune diseases, cardiovascular disorders, ophthalmic diseases, or asthma [
1]. Most approved mAbs are selected from three human IgG isotypes (IgG1, IgG2, or IgG4), which are defined by the total number of disulphide bridges (16 for IgG1 and IgG4, and 18 for IgG2), disulphide bonds in the flexible hinge region (two for IgG1 and IgG4, and four for IgG2) and the different heavy-chain amino acid sequences. At present, the majority of mAbs that have been approved by the FDA and EMA are based on the IgG1 isotype, while IgG3-based therapeutics have received little attention for therapy development owing to their short half-life in the body, given that the clearance rate of IgG3 is significantly faster than that of other isotypes [
2,
3]. As for the IgG4 isotype, there has been a progressive increase in the number of IgG4-based therapeutics on the market and in clinical trials. This is thanks to the remarkable progress that biotechnological and pharmaceutical companies have made in developing more stabilised IgG4 formats, such as by introducing S228P point mutation in the core-hinge sequence of the mAb [
2]. One of the therapeutic mAbs that is manufactured using hinge-stabilised engineering is nivolumab (Opdivo
® from Bristol-Myers Squibb, Dublin, Ireland), a human IgG4 mAb, which binds to the programmed death-1 (PD-1) receptor and blocks its interaction with the ligands PD-L1 and PD-L2, according to the Summary of Product Characteristics for Opdivo
® issued by the European Medicine Agency [
4]. By blocking this pathway, nivolumab potentiates T-cell responses, including anti-tumour responses [
5]. This is why this mAb is used, either by itself or in combination with another mAb, i.e., ipilimumab or cabozantinib, in cancer therapy. Opdivo
® is indicated for the treatment of melanoma; non-small cell lung cancer (NSCLC); malignant pleural mesothelioma (MPM); renal cell carcinoma (RCC); and classical Hodgkin lymphoma (cHL), among other types of cancer. Nivolumab is produced in Chinese Hamster Ovary (CHO) cells using recombinant DNA technology and is presented as a concentrate for solution for infusion (sterile concentrate) at 10 mg/mL [
4].
Like all mAbs, nivolumab consists of a large, highly complex molecule and is extremely sensitive to its environment [
6]. Before administration to patients, mAbs may be subject to environmental stress conditions such as exposure to light, and mechanical or thermal stresses, among others [
7,
8]. These stresses can have a negative impact on the structure of the protein, leading to its chemical or physical degradation [
9,
10,
11,
12]. Routine handling prior to administration—e.g., for sample preparation—or inadvertent mishandling—e.g., incorrect storage—can expose mAbs to these environmental stress conditions, so giving rise to the aforementioned forms of degradation [
8]. These forms of degradation alter the quality of mAb-based products and can have a significant effect on their therapeutic efficacy, by limiting their bioactivity or increasing their immunogenicity [
6,
7]. One of the main concerns with therapeutic proteins is the increase of aggregation in clinical samples, a form of degradation that could modify the ability of the mAb to correctly interact with its specific target, and could lead to immunogenicity. Detecting degraded mAbs before administration to patients is therefore crucial for ensuring high quality, efficacy, and safety [
7].
Forced degradation studies offer an opportunity to gain an in-depth understanding of the biophysical and biochemical properties of mAbs [
13]. They can provide valuable information on the degradation pathways to which mAbs are exposed during handling in hospitals prior to administration. Usually, these studies involve subjecting a particular biopharmaceutical product to a range of relatively harsh experimental stress conditions for a short period of time. It is important to avoid applying both too much or too little stress. The information that forced degradation studies provide is very useful in support of real-time environmental conditions and when dealing with practical problems, such as when there is a break in the cold chain; when making pharmaceutical preparations prior to administration; or when deciding whether to use the surplus product from one treatment on another patient [
13,
14,
15,
16]. The forced degradation conditions employed in such studies typically include high temperature; freeze-thaw cycles; agitation; light; high ionic strength exposure, among others. These conditions are selected according to the probability that the medicine may be exposed to them during the different stages of handling these products in hospital. The most common degradation pathways are aggregation, fragmentation, deamidation and oxidation [
13].
Forced degradation of nivolumab (Opdivo
®)—as a representative example of an IgG4—[
7] was conducted to evaluate the influence of degraded mAbs in a previously reported flow injection analysis (FIA) combined with UV spectroscopy and a statistical matching method [
17] for the quality control of compounded mAbs in hospital. Surprisingly, when this FIA strategy was initially proposed, it was not validated by checking the specificity, although the method was proposed for mAb quantification. As a result, the stress study conducted later on nivolumab focused more on validating the FIA methodology applied in the hospital quality control process than on presenting a detailed study of the degradation of nivolumab. It did not assess, for example, the functionality of degraded nivolumab. Within the context of a long-term stability study, nivolumab was studied in its original vials after opening and handling in a normal saline bag for intravenous infusion [
14]. Several physicochemical and functional properties of nivolumab were analysed. Although this research presented very valuable results, it did not focus on degradation studies, as its main objective was to study the stability of nivolumab over a period of more than 24 h, at different temperatures and after freeze/thaw cycles.
The aim of this research is therefore to carry out a comprehensive analytical characterisation and a forced degradation study of nivolumab (Opdivo® 10 mg/mL). For a proper characterisation of mAbs, complementary techniques must be used, including an appropriate set of physicochemical and functional tests. For this reason, we used a wide range of techniques to carry out this study, such as circular dichroism (CD) for the analysis of the secondary structure; fluorescence for the assessment of the tertiary structure; dynamic light scattering (DLS) to track particulate in the solutions; size-exclusion chromatography with UV detection (SE/UHPLC(UV)) to analyse oligomers; size-exclusion chromatography coupled to mass spectrometry (SE/UHPLC(UV)-MS(Orbitrap)) to determine the isoform profile including glycans; and an Enzyme-Linked Immunosorbent Assay (ELISA) to evaluate functionality by means of the specific bind nivolumab-PD1. To this end, forced degradation tests such as heating, freeze/thaw, agitation, light exposure and high ionic strength stresses were performed on samples of the medicine to evaluate degradation. These tests produced very interesting results that provide valuable new information about this biopharmaceutical, which could be useful for evaluating the potential consequences associated with its routine handling or unintentional mishandling in hospital.
2. Materials and Methods
2.1. Nivolumab Samples
Opdivo
® vials (nivolumab 10 mg/mL, Bristol-Myers Squibb Pharma EEIG, Dublin, Ireland) were kindly supplied for this study by the Pharmacy Unit of the University Hospital “San Cecilio” (Granada, Spain). Hospital leftovers were used throughout the study and the analyses were performed immediately after opening the vial to ensure the stability indicated by the manufacturer [
4]. The following batches were used during this study: ABJ9188; ABK3907; ABR9823; ABS8408; ABV0452; ABV2650; and ABW3154.
2.2. Forced Degradation (Stresses)
Forced degradation studies were carried out on the medicine Opdivo® by exposing the samples to each particular stress condition, always within the expiry date of the medicine once opened in order to avoid any degradation in nivolumab caused by the passage of time. Seven forced degradation conditions were tested: (i) exposure to a temperature of 40 °C for 1 h in a ThermoMixer® C thermoblock 1.5 mL (Eppendorf, Hamburg, Germany); (ii) exposure to a temperature of 60 °C for 1 h in a ThermoMixer® C thermoblock 1.5 mL (Eppendorf, Hamburg, Germany); (iii) one freeze–thaw cycle (room temperature of around 21/25 °C to −20 °C) in a vertical Bosch freezer (GSE32420, Gerlingen, Germany); (iv) five freeze–thaw cycles (room temperature of around 21/25 °C to −20 °C in a vertical Bosch freezer (GSE32420, Gerlingen, Germany); (v) exposure to light irradiation (250 W/m2) for 24 h in an aging chamber (Solarbox 3000e RH, Cofomegra, Milan, Italy); (vi) agitation (300 rounds/min) for 24 h in a mechanical laboratory shaker (type 3006, Gesellschaft für Labortechnik, Burgwedel, Germany); and (vii) exposure to a hypertonic medium by diluting in 1.5 M NaCl at a final concentration of 2 mg/mL.
Control samples were used throughout the study (samples of the medicine Opdivo® 10 mg/mL not subjected to stress and analysed within the first 24 h after opening the medicine vial while ensuring no sample degradation). Both control samples and those subjected to stress were analysed at the concentration used in the medicine (10 mg/mL), in order to avoid alterations such as displacement of the aggregation equilibrium by dilution. In the CD study, the analysis required the dilution of the samples in reverse-osmosis-quality water (purified with a Milli-RO plus Milli-Q station from Merk Millipore, Darmstadt, Germany) at a concentration of 0.1 mg/mL to avoid detector saturation. The NaCl stressed sample and its corresponding control sample had to be analysed at 2 mg/mL since NaCl was added as a concentrated aqueous solution. All the stress tests were performed using 200 µL of the medicine (Opdivo® 10 mg/mL), except for the high ionic strength test, for which a smaller volume of medicine was used. This was because a certain volume of NaCl had to be added to the sample at the right concentration to prepare a final volume of 200 µL. The temperature stresses were performed in 1.5 mL Eppendorf tubes (Eppendorf, Hamburg, Germany); the FTCs, agitation and high ionic strength tests were carried out in 2 mL amber RAM vials with a 9 mm thread (Symta, Madrid, Spain); and the light stress was performed in 2 mL clear RAM vials with a 9 mm thread (Symta, Madrid, Spain).
2.3. Physicochemical Analytical Methods
2.3.1. Visual Inspections
A quick visual inspection was carried out each day, prior to experimentation, in order to check for evidence of formation of large aggregates, turbidity, suspended particles, colour changes, and gas formation. This inspection was conducted using the naked eye and carried out by three different analysts.
2.3.2. Far Ultraviolet (UV) Circular Dichroism (CD) Spectroscopy
CD spectroscopy in the far region was used to study conformational changes in the nivolumab’s secondary structure over time. The experimental conditions were the same as those used in [
18]. Opdivo
® spectra were recorded using a JASCO J-815 spectropolarimeter (JASCO, Tokyo, Japan) equipped with a Peltier system for temperature control. Temperature was set at 20 °C for all measurements. The concentration of the solution samples to be analysed was optimized to 0.1 mg/mL of nivolumab. Spectra were acquired from 250 to 190 nm every 0.2 nm with a scan speed of 20 nm/min. The CD spectrum of NaCl stressed samples was acquired from 250 to 200 nm due to the fact that the high salt concentration increased the voltage to over 700 V at a wavelength of less than 200 nm, and the voltage should be less than 700 V to obtain reliable data [
19]. A total of five accumulations were averaged, with a bandwidth of 1 nm. A quartz cuvette with a path length of 1 mm was used. The first sample to be measured was the blank. The results for the blank were subtracted from all the samples so as to eliminate any possible interferences. Spectra Analysis software was used to apply the Means-Movement Smoothing to all the spectra. To determine the secondary structures, we used the Dichroweb website [
20] and the most suitable algorithm and DataSet were CONTIN [
21] and SP175 protein [
22], respectively. A control sample was also subjected to a temperature ramp from 20 °C to 90 °C.
2.3.3. Intrinsic Tryptophan Fluorescence Spectroscopy (IT-FS)
Conformational changes in the tertiary structure of nivolumab can take place even if the secondary structure remains unchanged. When the tertiary structure undergoes conformational changes, some hydrophobic pockets can be exposed to the solvent, which encourages the formation of aggregates. A Cary Eclipse spectrofluorometer (Agilent, Santa Clara, CA, USA) equipped with a Peltier system for temperature control was used to carry out IT-F measurements. The experimental conditions were similar to those used in [
18]. Emission spectra were recorded from 300 to 400 nm and the excitation wavelength was set at 298 nm. The samples were analysed at 10 mg/mL and the temperature was set at 20 °C. Excitation and emission slits were set to 2.5 nm and 5 nm, respectively. A total of five spectral accumulations were recorded for all measurements and the scan speed was set at 600 nm/min. The spectral centre of mass (C.M.) was considered as a mathematical representation of each spectrum, and was calculated using the following equation:
where λi is the wavelength and fi the fluorescence intensity.
2.3.4. Dynamic Light Scattering (DLS)
A Zetasizer Nano-ZS90 Malvern (Malvern Instruments Ltd. Worcestershire, UK) was used to assess soluble particulates (from 1 to 10,000 nm) in Opdivo
® (10 mg/mL nivolumab) samples. The experimental conditions were similar to those used in [
18]. A 1 cm spectrophotometry disposable cuvette was used to obtain the measurements. The temperature was set at 20 °C. A minimum of 100 reads were recorded per measurement and the acquisition time was 5 s per read. The average hydrodynamic diameter (HD), polydispersity index (PDI), polydispersity (Pd, %) and volume size distribution of all the samples were studied.
2.3.5. Isoform Analysis by Size-Exclusion Ultra-High-Performance Liquid Chromatography (UHPLC) with UV-Visible Detection Coupled to (Native) Mass Spectrometry (SE/UHPLC-UV-[Native] MS) Using Volatile Salts
The analysis was performed by liquid chromatography using a Dionex UltiMate 3000 chromatograph (Thermo Scientific, Waltham, MA, USA), equipped with two ternary bombs; a degasser; an autosampler; a thermostatic column compartment; and a multiple-wavelength detector (MWD-3000 Vis-UV detector). The chromatograph was coupled in line to a Q Exactive™ Plus Hybrid Quadrupole Orbitrap mass spectrometer (Thermo Scientific). The ionisation was performed using a heated electrospray ionisation (HESI) source. Chromatographic and MS conditions were similar to those used in [
23]. A SEC column 300 A, 2.7 μm, 4.6 × 300 mm (AdvanceBioSec, Agilent technologies, Santa Clara, CA, USA) was used and 8 µg of Opdivo
® was injected into the column. The column temperature was set at 30 °C. This analysis was carried out under isocratic conditions of 100 mM ammonium acetate buffer (LC-MS purity grade, Sigma Aldrich, Madrid, Spain) prepared in reverse-osmosis-quality water, at 0.3 mL/min for 20 min. The UV chromatograms were registered at different wavelengths, i.e., λ= 214 nm, λ= 220 nm and λ = 280 nm, using a bandwidth of 5 nm in all cases. The MS method was carried out in full positive polarity mode at a resolution of 17,500 (defined at
m/z 200). The mass range was set at 2500–6000
m/
z and the automatic gain control (AGC) target value was 3.0 × 10
6 with a maximum injection time of 200 ms and 10 microscans. In-source collision-induced dissociation (CID) was set to 100 eV. The MS instrumental parameters were set as follows: the sheath gas flow rate was 20 arbitrary units (AU); auxiliary gas flow rate was 5 AU; spray voltage was 3.6 kV; capillary temperature was 275 °C; probe heater temperature was 275 °C; and S-lens RF voltage was 100 V.
The analysis was performed by liquid chromatography using a Dionex UltiMate 3000 chromatograph (Thermo Scientific, Waltham, MA, USA), equipped with two ternary bombs; a degasser; an autosampler; a thermostatic column compartment; and a multiple-wavelength detector (MWD-3000 Vis-UV detector). The chromatograph was coupled in line to a Q Exactive™ Plus Hybrid Quadrupole Orbitrap mass spectrometer (Thermo Scientific). The ionisation was performed using a heated electrospray ionisation (HESI) source. Chromatographic and MS conditions were similar to those used in [
23]. A SEC column 300 A, 2.7μm, 4.6 × 300mm (AdvanceBioSec, Agilent technologies, Santa Clara, CA, USA) was used and 8 µg of Opdivo
® was injected into the column. The column temperature was set at 30 °C. This analysis was carried out under isocratic conditions of 100 mM ammonium acetate buffer (LC-MS purity grade, Sigma Aldrich, Madrid, Spain) prepared in reverse-osmosis-quality water, at 0.3 mL/min for 20 min. The UV chromatograms were registered at different wavelengths, i.e., λ= 214 nm, λ= 220 nm and λ = 280 nm, using a bandwidth of 5 nm in all cases. The MS method was carried out in full positive polarity mode at a resolution of 17,500 (defined at
m/z 200). The mass range was set at 2500–6000
m/
z and the automatic gain control (AGC) target value was 3.0 × 10
6 with a maximum injection time of 200 ms and 10 microscans. In-source collision-induced dissociation (CID) was set to 100 eV. The MS instrumental parameters were set as follows: the sheath gas flow rate was 20 arbitrary units (AU); auxiliary gas flow rate was 5 AU; spray voltage was 3.6 kV; capillary temperature was 275 °C; probe heater temperature was 275 °C; and S-lens RF voltage was 100 V.
Native isoforms of the medicine and stressed samples were obtained by mass spectra deconvolution using BioPharma Finder™ software version 3.1 (Thermo Scientific) with ReSpect algorithms.
2.3.6. Aggregation Study by Size-Exclusion Ultra-High-Performance Liquid Chromatography (UHPLC) with UV-Visible Detection (SE/UHPLC-UV) Using Non-Volatile Salts
The aggregation study was conducted by liquid chromatography using the chromatograph (Dionex UltiMate 3000 chromatograph, Thermo Scientific) and SEC column (300 A, 2.7 μm, 4.6 × 300mm, AdvanceBioSec) described above. In this particular analysis, the chromatograph was used separately from the Q Exactive™ Plus Hybrid Quadrupole Orbitrap mass spectrometer. Therefore, the mobile phase was composed of non-volatile salts: 150 mM of phosphate buffer pH 7.0, which was prepared with monohydrate monobasic sodium phosphate (Panreac, Barcelona, Spain) and anhydrous disodium hydrogen phosphate (Panreac, Barcelona, Spain) in reverse-osmosis-quality water. The analysis was performed under isocratic conditions for 18 min. The column temperature was set at 30 °C and 8 µg of sample (Opdivo®) were injected into the column at 0.3 mL/min. The UV chromatograms were registered at 214 nm, 220 nm and 280 nm, using a bandwidth of 5 nm in all cases.
The SEC column was calibrated to establish the relationship between the molecular weight and the retention time of nivolumab. The calibration kit (Agilent, Santa Clara, CA, USA) was composed of five proteins: thyroglobulin (670 kDa), γ-globulin (150 kDa), ovalbumin (45 kDa), myoglobin (17 kDa) and angiotensin II (1 kDa), although this last protein was not used in the calibration of the SEC column, due to its low molecular weight (M.W.). The experimental size exclusion column calibration model is M.W. = 1 × 108e−0.828x; R2 = 0.9989.
2.4. Functional-Based Method: Enzyme-Linked Immunosorbent Assay (ELISA)
In order to test the biological activity of nivolumab, we developed and optimised a new indirect, non-competitive ELISA method on the basis of the available bibliography [
24,
25,
26] and of our previous research into other therapeutic mAbs [
18,
27,
28]. The optimised method was as follows: firstly, 96-well Maxisorp immune plates were sensitised by adding 100 μL/well of 0.5 μg/mL Human PD-1 (CD279), FC Fusion (Sigma Aldrich, Madrid, Spain) diluted in 0.1 M carbonate buffer solution pH 9.6 (prepared with sodium carbonate (Panreac, Spain) and sodium bicarbonate (Panreac, Spain)) and incubated overnight (18 h) at 4 °C. The plates were manually washed four times with 200 μL/well of PBS-Tween 20 pH 7.4 containing 0.3% (
v/
v) Tween 20
®. PBS was prepared using sodium chloride, potassium chloride, disodium phosphate monohydrate and potassium phosphate monobasic supplied by Panreac (Barcelona, Spain), while Tween 20 was supplied by Guinama (Valencia, Spain). The plates were then treated with 200 μL/well of the blocking buffer (PBS-Tween 20 pH 7.4 containing skimmed milk 2% (
w/
v)) for 2 h at 37 °C to eliminate nonspecific absorptions. After that they were washed four more times and filled with a 100 μL/well of nivolumab appropriately diluted in 0.1 M carbonate buffer (pH 9.6) at three concentrations: 5 ng/mL, 10 ng/mL and 25 ng/mL. The plates were incubated at 37 °C for 45 min in a universal digitronic precision oven P Selecta
® (J.P. Selecta, s.a. Abrera, Barcelona, Spain), after which they were washed four times with PBS-Tween 20 and incubated again with 100 μL/well of 1:1000 in PBS diluted anti-human IgG4-HRP (mouse anti-human IgG4 Fc antibody-HRP conjugate, Thermo Fisher, Landsmeer, the Netherlands) for 30 min at 37 °C. After being washed for four times, 100 μL/well of the substrate solution (O-Phenylenediamine Dihydrochloride (OPD), Sigma Aldrich, Madrid, Spain) were added and incubated for 20 min at room temperature (around 25 °C) in darkness. Finally, 50 μL/well of 1M sulfuric acid solution was added to stop the reaction. Absorbance was recorded at 450 nm and 620 nm, and the analytical signal was the difference between the two absorbance values (NanoQuant Infinite 200 Pro, Tecan, Austria, GMBH).
This ELISA format allowed us to assess the specific interaction between nivolumab and its target, the PD-1 receptor. In this way, we were able to evaluate and quantify the biological activity of nivolumab after applying the stress factors indicated previously. To this end, the method was validated in terms of the calibration model, precision and accuracy.
The next stage was to investigate the standard calibration model using standard samples of nivolumab. In this case, fresh samples of the medicine Opdivo
® (10 mg/mL nivolumab) were used as the standard due to the lack of any proper standard samples of nivolumab. The nivolumab concentrations checked to obtain the calibration model were 100, 50, 25, 10, 5, 1, and 0.5 ng/mL diluted in 0.1 M carbonate buffer (pH 9.6). Each standard concentration was prepared and analysed in quintuplicate. From the graphical distribution results (absorbance vs. ng/mL nivolumab), the selected optimum concentrations for analysing nivolumab were 25, 10 and 5 ng/mL, as the corresponding response values were widely distributed within the permitted absorbance range, i.e., from 0 to 1. The Statgraphics Centurion 18 (Statgraphics Technologies, The Plains, VA, USA) software package was used for the statistical analysis of the calibration models. The coefficient of determination (R
2) and the lack-of-fit signification test were used to evaluate the best fit of these data to the different mathematical models. Once these concentrations were selected as representative of the calibration model, the precision and the accuracy of this ELISA method were evaluated according to the ICH recommendations Q2(R1) at these three concentrations [
29]. The precision was checked as repeatability (intraday precision) and intermediate precision (interday precision; over three consecutive days). For the repeatability study, nine standard solutions were prepared within the same day and under the same experimental conditions: 3 samples at 5 ng/mL, 3 samples at 10 ng/mL and 3 samples at 25 ng/mL. Intermediate precision was assessed by analysing three standard solutions (5, 10 and 25 ng/mL of nivolumab) that were freshly prepared on three different days and under the same experimental conditions. Both repeatability and intermediate precision were determined as the relative standard deviation (RSD, %) of the concentrations calculated from the absorbance using the standard calibration curve. Accuracy was assessed by analysing three standard solutions of each concentration of nivolumab and determining the mean recovery percentage (R, %) for each concentration.
The functional study of the stressed samples was performed by simultaneously analysing control (fresh) and stressed nivolumab samples (both from Opdivo®) using the validated ELISA method. The results were then statistically compared (Student’s t analysis with 95 % of confidence) to find out whether the ability of nivolumab to specifically bind to its target, the PD-1, had been altered in any way. To this end, the biological activity of each of the stressed samples was compared with that of the control sample. The reported biological activity was the average of three replicates.
4. Discussion
Throughout this study, we used samples of nivolumab in its medicine formulation (Opdivo®, 10 mg/mL). We began by characterising fresh control samples of nivolumab, which were then compared with stressed samples that had been subjected to a range of different stress conditions. The aim of these tests was to detect and identify possible changes in the medicine such as aggregation, denaturation or structural modifications, and the appearance of new particulates.
Results indicate that nivolumab medicine (Opdivo
®, 10 mg/mL) is composed of monomers and natural dimers in relative proportions of 99.6% and 0.4%, respectively, as highlighted in the SEC profiles conducted on the batches (
Table 6). The native LC-MS(Orbitrap) isoform profile indicated three main glycoforms, which were assigned to A2G0F/A2G0F, A2G0F/A2G1F and A2G1F/A2G1F, in accordance with their particular masses (
Figure 4 and
Table 5). As regards the secondary structure of nivolumab derived from the CD spectrum, this consisted mainly of β-sheet (
Table 2), as is normal in mAbs [
18,
27], and showed the characteristic spectral parameters: minimum at around 218.0 nm, shoulder at 228.7 nm and wavelength for ellipticity = 0 of around 209.7 nm. The tertiary structure, which was characterised by IT-FS, showed a centre of spectral mass (C.M.) of 361 nm (
Table 3) for both the undiluted (10 mg/mL) and diluted (2 mg/mL) medicine. The DLS study indicated an HD of around 9.7 nm and 9.3 nm and a polydispersity of 11.7% and 12.4% for the undiluted nivolumab samples used as control, while the diluted medicine showed similar HD (9.4 nm) and polydispersity (7.4%) (
Table 4). All these values are shared with other therapeutic mAbs [
18,
27].
The heat stress study was carried out by subjecting the medicine (Opdivo
®, 10 mg/mL) to two different temperatures, 40 °C and 60 °C, for 1 h. In both cases, there were no significant changes in the medicine. SE/UHPLC(UV) analysis did not show any increase in the dimer population or the appearance of high molecular weight (HMW) aggregates (
Figure 5). The lack of aggregation was also corroborated by DLS, which only detected one population of particulate, i.e., the monomers. However, in the sample subjected to 60 °C, there was an increase in HD (from 9.7 ± 3.2 nm in the control to 11.7 ± 5.8 nm in the stressed sample) and PDI (from 0.21 in the control to 0.32 in the stressed sample), which could be attributed to an increased hydration of the nivolumab monomers; this could be due to the fact that the heat stress temperature (60 °C) was very close to the temperature beyond which the secondary structure began to deteriorate (65 °C) (
Table 4). In addition, CD and IT-FS did not detect any changes in the secondary and tertiary structures of the protein (
Table 1 and
Table 2). For its part, the isoform profile was very stable and the glycoforms remained unaltered. The same glycoforms, with similar relative abundances, were identified in both the heat-stressed and control samples of nivolumab (
Figure 4 and
Table 5). Moreover, ELISA assays demonstrated that subjecting nivolumab to 40 °C and 60 °C for 1 h did not affect its functionality, as measured in terms of its capacity to bind to its target (the PD-1) (
Figure 6). Although previous researchers reported slight structural changes in IgG4 [
35] and in nivolumab [
7] when subjected to more aggressive heat stress, in this study we have demonstrated that nivolumab (Opdivo
® 10 mg/mL) remains stable when subjected up to 60 °C for 1 h, and its binding capacity to PD-1 is unaffected.
It is well known that freeze/thaw cycles (FTC) can lead to protein aggregation, as they disturb the local structure on the surface of the residues of the protein, so giving rise to partial denaturation during freezing, which culminates in an aggregation process [
36]. Nevertheless, no changes were noted in nivolumab in either FTC 1 or FTC 5. In both cases, the secondary and tertiary structures remained stable, as did the relative proportions of monomers and dimers. Moreover, DLS confirmed the absence of aggregates and the isoform profile remained unaltered. ELISA results showed that the nivolumab-PD-1 binding capacity decreased slightly to around 90% after one and five cycles. This means that this functional property is slightly affected by the freeze/thaw cycles, even though none of the physicochemical parameters that we studied indicated modifications in the medicine (
Figure 6). As indicated earlier, this result is in line with the instruction in the technical report on Opdivo
® [
4] about not freezing this medicine.
Agitation for 24 h had no significant effects on the particulate in the nivolumab sample. SE/UHPLC(UV) demonstrated that there were no changes in the relative proportions of monomers and dimers, and DLS also confirmed that no new populations or aggregates appeared after application of this stress. In addition, the CD results showed a similar spectrum to the control sample, so indicating that the secondary structure remained unchanged. The same occurred with the tertiary structure, given that the IT-FS results for the stressed sample had a similar profile to the control, and their C.M. values were exactly the same. The isoform profile also remained unchanged, as revealed by the fact that the glycoforms assigned to the stressed sample matched those assigned to the control one. However, ELISA indicated a reduction in the capacity of nivolumab to bind to PD-1 (
Figure 6 and
Table 9), which could not be due to any structural modification, given the aforementioned results. However, it could be due to changes in the tertiary structure of nivolumab, given that the maximum emission of tryptophans in nivolumab is over 350 nm, which is a very high value, similar to that of the amino acid in aqueous solution [
37]. This indicates that the tryptophans in nivolumab are close to a hydrophilic environment, and are therefore highly exposed to the solvent. As a result, IT-FS is unable to detect the structural changes that might lead to the loss of biological activity revealed by ELISA. In the near future, we will be investigating these findings in much greater detail using peptide mapping analysis.
As regards the stress involving exposure to light for 24 h, non-natural aggregation was observed by SE/UHPLC(UV) (
Figure 5). Previous research on other mAbs has shown that exposure to light induces aggregation [
30,
38], a finding that was also true for nivolumab medicine samples, in which the SE-chromatographic profile showed an increase in the peak for dimers, whose relative proportion was 3% higher than in the control sample (
Table 6). Despite the increased proportion of dimers, CD assessment did not detect any changes in the secondary structure of nivolumab. As regards the tertiary structure of the protein, changes were detected, not in the C.M. (indicating no protein unfolding) of the IT-FS spectra, but in their intensity. This was probably due to the light-triggered oxidation of tryptophan residues. These changes may have caused the loss of biological activity, which could also have been induced by the aggregation of mAbs, as reported in previous research [
39]. As expected, the nivolumab sample exposed to light showed a statistically highly significant decrease in the binding capacity of nivolumab to PD-1 (
Figure 6), falling to around 70% of that of the control sample (
Table 9). The isoforms profile was also very different because of the chemical changes (i.e., oxidations) caused by light exposure, which modified the molecular weight of nivolumab. This shows that the isoform profile was greatly affected by exposure to light despite being unaffected by the other forms of stress (
Figure 4). Two new nivolumab isoforms were detected after the light exposure test with different masses compared to those detected in the control sample. No N-glycoforms could be attributed to these isoforms. All these results clearly indicated that exposure to light stress caused important degradation of nivolumab. These changes were probably due to the oxidative process to which the sample is exposed, as many previous publications suggest. We obtained similar results in a parallel study of nivolumab (manuscript in preparation).
As regards the stress by exposure to high ionic strength, the SE/UHPLC(UV) and DLS results indicated that no aggregation occurred, as monomers and dimers appeared in the same proportions as in the control sample and no HMW aggregates were detected, either by SEC or by DLS. Nevertheless, DLS results showed an increase in the HD value (from 9.4 ± 2.6 nm in the control to 13.9 ± 3.8 nm in the stressed sample) and in the PDI (from 0.29 in the control to 0.34 in the stressed sample). This was attributed to an increase in hydration, as indicated in
Section 3.4 DLS (
Table 4). CD results indicated that the secondary structure of nivolumab was not affected by this stress (
Table 3). Despite all these results, ELISA indicated two significant values in
Figure 6, referring that the binding capacity of nivolumab to the PD1 slightly (and significantly) decreased (around a 10%) with respect to the control samples. This could be due to the proposed increase in the hydration of the surface of the protein due to the increase in ionic strength, which might affect the capacity of nivolumab to bind to PD-1, its therapeutic target. The changes detected by DLS may therefore contribute to this loss of biological activity.
5. Conclusions
Biopharmaceuticals can be exposed to a wide range of environmental stress conditions during routine handling in hospitals, as well as in previous stages such as research, development and manufacturing. The research findings presented in this paper focus above stresses to which nivolumab (Opdivo®) might be subject when handled in hospital prior to administration. To assess the impact of these stresses, various stress tests were performed. The results of these tests indicate that nivolumab is most affected by accelerated exposure to light. This is because light causes non-natural aggregation (dimerisation) of the protein and promotes oxidations (i.e., tryptophan residues). As a consequence of these chemical changes, it also alters the isoform profile. The results of the functional studies, which measured the capacity of nivolumab to bind to the PD-1 receptor, demonstrated that this capacity is substantially reduced when nivolumab is subject to stress by exposure to light. This means that it should be stored and handled away from the light so as to avoid degradation. On the other hand, nivolumab (Opdivo®) was found to remain highly stable when subjected to heat exposure for 1 h up to 60 °C, even though this was very close to the temperature at which nivolumab starts to deteriorate due to heat stress. As regards the agitation test, even though no significant conformational changes, aggregation or particulate were observed, the biological activity of nivolumab was affected, in that it caused a decrease in the ability of nivolumab to bind to the PD-1 receptor (estimated at around 20%). We therefore strongly recommend, as far as possible, to avoid shaking the medicine and its pharmaceutical preparations and to take great care during transport and handling. Caution must also be taken when diluting the medicine in NaCl, as physical modifications were detected when nivolumab was exposed to a highly hypertonic solution. We have also confirmed that freezing/thawing could affect the capacity of nivolumab to bind to PD-1, a finding that confirms the instruction in the Opdivo® technical report about not freezing the medicine. As expected, this research highlights the fragile nature of biopharmaceutics and, in particular, of nivolumab.