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An intrinsic fluorescence method for the determination of protein concentration in vaccines containing aluminum salt adjuvants
Vaccine xxx (2018) xxx–xxx
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An intrinsic fluorescence method for the determination of protein concentration in vaccines containing aluminum salt adjuvants Lucienne Nouchikian 1, Cristopher Roque, Jimmy Y. Song 2, Nausheen Rahman, Salvador F. Ausar ⇑ BioProcess Research and Development, Sanofi Pasteur, 1755 Steeles Ave West, Toronto, Ontario M3R 3T4, Canada
a r t i c l e
i n f o
Article history: Received 20 April 2018 Received in revised form 12 July 2018 Accepted 3 August 2018 Available online xxxx Keywords: Intrinsic fluorescence Adjuvants Aluminum salt adjuvant Vaccines Protein quantification
a b s t r a c t Determination of protein concentration in vaccines containing aluminum salt adjuvant typically necessitates desorption of the protein prior to analysis. Here we describe a method based on the intrinsic fluorescence of tyrosine and tryptophan that requires no desorption of proteins. Adjuvanted formulations of three model Bordetella pertussis antigens were excited at 280 nm and their emission spectra collected from 290 to 400 nm. Emission spectra of protein antigens in the presence of aluminum salt adjuvants were able to be detected, the effects of adjuvants on the spectra were analyzed, and linear regressions were calculated. The fluorescence method proved to be very sensitive with a limit of quantification between 0.4 and 4.4 mg/mL and limit of linearity between 100 and 200 mg/mL, across the formulations tested. The fluorescence method was found to be influenced by adjuvant presence, type of adjuvant, adjuvant concentration, buffer and pH conditions. The method also demonstrated ability to monitor the percent adsorption of antigens to the adjuvants. Furthermore, intrinsic fluorescence showed good correlation with micro-Kjeldahl elemental assay in quantifying protein concentration. Being a non-invasive, quick and sensitive method, intrinsic fluorescence has the potential to be utilized as a high throughput tool for vaccine development and conceivably implemented in-line, using in-line fluorimeters, to monitor antigen concentration during formulation processing. Ó 2018 Published by Elsevier Ltd.
1. Introduction The concentration of antigens in vaccine products is an important parameter that needs to be controlled to ensure consistency in the manufacturing process and product quality. Monitoring the concentration of antigens during processing is important for several reasons such as controlling formulation accuracy, estimating process-related losses, monitoring product homogeneity, and ensuring appropriate adsorption of antigens to a given adjuvant [1,2]. Typically protein-based vaccines are presented as suspensions or emulsions due to the presence of adjuvants such as aluminum salts (i.e., AlOOH, AlPO4, etc.), or squalene emulsions (i.e., squalene AS03 emulsion, MF59, etc.). Adjuvants are a key component in vaccines [3–5]; however, the presence of adjuvants can produce significant interference with commonly used methods for protein ⇑ Corresponding author. E-mail address: [email protected] (S.F. Ausar). Present address: Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada. 2 Present address: Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66047, USA. 1
quantification, due to turbidity introduced by the adjuvant particles. For the case of vaccines containing aluminum salts, many methods have been published for analysis of antigens, but they necessitate antigen desorption prior to testing due to the turbidity of the formulations [6–8]. Desorption procedures described in the literature are rather complex and time-consuming, with variable recovery and questionable reproducibility [9,10]. Other methods reported for the quantification of antigens in adjuvanted vaccines, such as, flow cytometry [11], chemiluminescent nitrogen detection [12], micro-kjeldalh [13] and o-phthalaldehyde assay [9], can be directly applied to suspensions. However, these methods are mostly destructive, requiring specific antibodies or special reagents, and are therefore not suitable for fast turnaround or in-line process monitoring. Proteins are unique among biomolecules in displaying intrinsic fluorescence (IF) due to the presence of aromatic amino acids. Most proteins contain aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe). Upon excitation in the UV region (250 nm for Phe, 275 nm Tyr and 290 nm for Trp), these aromatic amino acids can produce an emission spectrum with maximum emission in the range of 310–360 nm [14], in which the intensity is proportional to the protein concentration.
https://doi.org/10.1016/j.vaccine.2018.08.005 0264-410X/Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Nouchikian L et al. An intrinsic fluorescence method for the determination of protein concentration in vaccines containing aluminum salt adjuvants. Vaccine (2018), https://doi.org/10.1016/j.vaccine.2018.08.005
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Amongst the three fluorescent amino acids found in proteins, Phe contribution to the IF of protein is negligible due to the low quantum yield. Although Trp and Tyr possess similar quantum yields, the indole group of Trp has been reported to be the dominant source of UV absorbance near 280 nm and fluorescence emission near 330 nm in proteins [15]. Despite the relatively inferior fluorescence properties of Tyr, analysis of its IF has been shown to be a useful tool for analysis of proteins that are lacking Trp [16]. Many reports in the literature have described the use of IF, especially the use of Trp, to study changes in emission wavelength and intensity, in order to analyze conformational changes in experimental vaccines containing toxoids [17], inactivated [18] and attenuated viruses [19], virus like particles [20], and aluminum salt adsorbed proteins [21,22]. However, the use of IF to monitor protein concentration has not been well explored. Previous research from our laboratory has indicated that it is possible to detect the fluorescence of extrinsically added dyes to formulations containing aluminum salt adjuvants with no detectable adjuvant interference [23]. Based on that premise, we hypothesize that the aluminum salts would not significantly interfere with the IF of proteins, and it could serve as a method for protein quantification in a matrix of mineral salt adjuvants. This is due to the fact that fluorescence is collected at a 90° angle, which reduces the inner filtering effects from sample turbidity [24]. In this paper we use the Bordetella pertussis antigens Filamentous haemagglutinin (FHA), Pertactin (PRN) and Fimbria (FIM), to demonstrate that IF of proteins can be detected in formulations containing aluminum salts allowing the development of a rapid, simple and non-destructive method of quantifying antigen concentration in vaccine formulations. The method was developed for antigens formulated in aluminum phosphate (AlPO4) and aluminum oxyhydroxide (AlOOH), as these are the most commonly used aluminum salts in vaccines.
then mixed on an AdamsTM Nutator Mixer (Becton Dickinson, New Jersey, United States) at room temperature for 30 min to allow for antigen adsorption to the adjuvant. Preliminary experiments indicated that the percentage adsorption does not change significantly after 30 minutes (min) of mixing with the adjuvant. Unless otherwise stated, the buffer used for the standard curves and test samples was TBS. However, other buffers (PBS, HEPES, citrate, and bicarbonate) were used to test the effect of buffer and pH conditions on the calibration curves. To test the effect of percent adsorption, different concentrations of phosphate (0 mM, 2 mM, 20 mM, and 80 mM) were added to the formulations in TBS. Test samples containing FHA, prepared to assess the effect of adjuvant presence and inter-assay variation on calibration curve accuracy, were made at antigen concentrations of 10, 50 and 200 mg/mL, with an adjuvant concentration of 0.66 mg Al/mL for the formulations containing either AlOOH or AlPO4. Test samples containing FHA, created to assess the effect of sedimentation, were prepared at an antigen concentration of 100 mg/mL, with 0.66 mg Al/mL adjuvant concentration for the formulations containing either AlOOH or AlPO4. Test samples containing PRN, created to assess the effect of adjuvant presence on calibration curve accuracy, were prepared at antigen concentrations of 10, 50 and 100 mg/mL, with an adjuvant concentration of 0.66 mg Al/mL for the formulations containing either AlOOH or AlPO4. Test samples containing PRN, created to assess the effect of buffer and pH on calibration curve prediction accuracy, were prepared at an antigen concentration of 100 mg/mL, with an adjuvant concentration of 0.66 mg Al/mL for the formulations containing either AlOOH or AlPO4. Test samples containing FIM, prepared to assess the effect of adjuvant presence on calibration curve accuracy, were prepared at antigen concentrations of 10, 50 and 200 mg/mL, with an adjuvant concentration of 0.66 mg Al/mL for the formulations containing either AlOOH or AlPO4.
2. Materials and methods 2.3. Fluorescence measurements 2.1. Materials Filamentous haemagglutinin (FHA), molecular weight (MW) = 220 kDa, pI = 8.70; Pertactin (PRN), MW = 60.3 kDa, pI = 6.03; Fimbriae (FIM), type 2, MW = 19.2 kDa; pI = 5.94, and type 3, MW = 17.2 kDa, pI = 9.13, were manufactured and provided by Sanofi Pasteur (Toronto, Canada). With respect to aromatic amino acids residues relevant to IF, it is important to mention that FHA has 11 tryptophan and 28 tyrosine, PRN has 8 tryptophan and 6 tyrosine, FIM Type 2 has no tryptophan and 8 tyrosine, and FIM Type 3 has no tryptophan and 7 tyrosine. Batches of FHA, PRN and FIM were prepared at concentrations ranging from 973.4–1200.9 mg/mL, 147.7–241.9 mg/mL and 242. 0–402.0 mg/mL, respectively. The AlOOH adjuvant was supplied at 10.2 mg Al/mL by Brenntag Biosector (Frederikssund, Denmark). The AlPO4 adjuvant was prepared by Sanofi Pasteur (Toronto, Canada) at 4.1 mg Al/mL. The buffers 10 mM Tris-HCl with 150 mM NaCl, pH 7.4 (TBS); phosphate buffered saline (PBS); 150 mM citrate, pH 6; 100 mM bicarbonate, pH 9; and 0.5 M phosphate, pH 7.4; were made in house with reagents supplied by Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). HEPES was supplied by Life Technologies at 1 M concentrated solution and diluted to 10 mM with Milli-Q water and the pH was adjusted using 1 M HCl. 2.2. Preparation of formulations All formulations were prepared by adding the appropriate amount of adjuvant to the applicable amount of protein, and then using the designated buffer as a final diluent. Formulations were
Fluorescence measurements of all formulations were collected using Chirascan Plus spectrophotometer (Applied Photophysics, Surrey, United Kingdom). The samples were excited at an excitation wavelength of 280 nm and the absolute fluorescence intensities were collected between 290 and 400 nm, at 1 nm increments and a measurement time per point of 0.5 s, using a constant photomultiplier tube setting of 800 V and a set temperature of 20 °C. Once a maximum emission peak was found, the spectra collection was set to ±20 nm from the maximum emission peak. Standard 10 mm square quartz cuvettes were used to measure the formulations. The use of square geometry cuvettes provided better spectral quality when compared to the triangular front-face counterpart (data not shown). Readings were taken immediately after mixing for optimal results. 2.4. Sedimentation study FHA samples were mixed by 10 repeated inversions, and the fluorescence intensity from 290 to 400 nm was collected at 2 min intervals, for 20 min. The fluorescence intensity, at 337 nm, was averaged between the duplicates. 2.5. Calibration curves Calibration curves were created in duplicate following International Standard ISO 11095 [25]. The FHA calibration curves created to assess the effect of adjuvant were made with samples at antigen concentrations of 0, 1, 5, 10, 50, 100 and 200 mg/mL, and an adjuvant concentration of 0.66 mg Al/mL for the formulations
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containing either AlOOH or AlPO4. The FHA calibration curves created to assess the effect of adjuvant concentration were made with samples at antigen concentrations of 5, 10, 50, 100 and 200 mg/mL, and AlOOH or AlPO4 concentrations of 0.33 mg Al/mL, 0.66 mg Al/ mL and 1.32 mg Al/mL. The PRN calibration curves created to assess the effect of adjuvant were made with samples at antigen concentrations of 0, 1, 5, 10, 50 and 100 mg/mL, and an adjuvant concentration of 0.66 mg Al/mL for the formulations containing either AlOOH or AlPO4. The PRN calibration curves created to assess the effect of buffer composition and pH, were made with samples at antigen concentrations of 0, 5, 10, 50 and 100 mg/mL, and an AlOOH concentration of 0.66 mg Al/mL. The FIM calibration curves created to assess the effect of adjuvant were made with samples at antigen concentrations of 0, 1, 5, 10, 50, 100, 150 and 200 mg/mL, and an adjuvant concentration of 0.66 mg Al/mL for the formulations containing either AlOOH or AlPO4. The wavelength at which the maximum intensity was reached was selected and the fluorescence at this point was averaged between duplicates and plotted against its concentration. For the calibration curves generated to assess the effect of adjuvant and adjuvant concentration, the fluorescence intensities were averaged between the duplicates. For all other calibration curves, no replicates were obtained. Limit of detection (LOD) and limit of quantification (LOQ) were calculated using the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guide lines [26], as follows: LOD = 3.3r/S LOQ = 10r/S r = standard deviation of the blank S = slope of the linear regression curve. Limit of linearity (LOL) was established by removing data points, starting from the highest concentration of antigen, from the calibration curve, until both the R2 of the linear regression was 0.980 and the runs test for departure from linearity produced a p-value > 0.05, using GraphPad Prism version 6.00 (GraphPad Software, La Jolla California, USA). Statistical comparison of test samples and linear regression slopes were performed by analysis of variance (ANOVA) and analysis of covariance (ANCOVA), respectively, using GraphPad Prism version 7.02 (GraphPad Software, La Jolla California, USA). Test samples and slopes were considered to be statistically different if the probability (p-value) of obtaining a test statistic, F, greater than the one calculated by pure chance was valued at 0.05 or less. 2.6. Inter-assay variability Inter-assay variability was investigated by measuring 10 aliquots of test samples, prepared at low (10 mg/mL), medium (50 mg/mL) and high (200 mg/mL) concentrations of FHA adsorbed to 0.66 mg Al/mL AlOOH, against 10 aliquoted calibration curves of FHA-AlOOH formulations. 2.7. Percent adsorption Percent adsorption was tested with 50 mg/mL of PRN adsorbed to 0.66 mg Al/mL AlOOH with the presence of 0 mM, 2 mM, 20 mM and 80 mM phosphate, to create a difference in the percent adsorption levels. The fluorescence of the total formulations was collected, along with the fluorescence of the supernatant, after centrifugation at 20,000 x g for 10 min on an Eppendorf 5417 centrifuge (Eppendorf, Germany). The concentration of the
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formulations, and the supernatant, was back-calculated using their respective standard curves. 2.8. Comparison with micro-Kjeldahl Samples of unknown antigen concentrations were measured by both micro-Kjeldahl [27] and IF methods. For the IF method, adjuvant free samples were diluted in TBS, and samples containing AlPO4 were diluted in TBS containing 0.66 mg Al/mL of AlPO4, until a concentration value within the respective calibration curve range was obtained. All dilutions were back calculated using the linear regression of their respective standard curve. The back calculated concentrations were compared against the micro-Kjeldahl results. 3. Results 3.1. Effect of aluminum salt adjuvants on the fluorescence spectrum of protein antigens To investigate the feasibility of the IF method for the detection of protein concentration in the presence of aluminum salt adjuvants, formulations of FHA, PRN and FIM, at 100 mg/mL, in the presence of AlOOH or AlPO4, were excited at 280 nm and their full emission spectra from 290 to 400 nm was immediately collected in square 10 mm cuvettes (Fig. 1). In the absence of aluminum salts, both FHA and PRN showed broad and well detectable peaks, with a maximum at 337 nm ± 1 nm (n = 3), (Fig. 1A and B), while FIM displayed a narrower peak with maximum fluorescence intensity at 310 nm ± 0 nm (n = 3) (Fig. 1C). The lower emission wavelength detected in FIM is attributed to a lack of tryptophan in its structure, making tyrosine the primary contributor to the fluorescence spectrum [28]. The addition of aluminum salts did not significantly affect the maximum emission wavelength. However, the fluorescence intensity was impacted depending on the type of adjuvant used in the formulation. While a significant increase in the fluorescence intensity was observed for AlPO4 containing samples, the presence of AlOOH did not notably impact the fluorescence spectra of the proteins under investigation (Fig. 1). To investigate any potential time-dependent structural changes that might influence the fluorescence signals during data acquisition, the peak position of the Trp fluorescence of FHA as a function of time was analyzed. No noticeable changes were observed on the peak position of the fluorescence spectra of FHA for up to 20 min of incubation at room temperature (Supplementary Fig. 1). Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.vaccine.2018.08. 005. Altogether, the results indicate that the presence of aluminum salts at adjuvant concentrations commonly used in vaccines does not interfere with the fluorescence properties of proteins, making this method feasible for the detection of proteins in aluminum salt adjuvanted vaccines. 3.2. Effect of sedimentation Suspensions of aluminum salt adjuvants are known to sediment over time [29]. To investigate the effect of sedimentation on the IF, the fluorescence intensity of formulations containing 100 mg/mL FHA alone, or adsorbed to 0.66 mg Al/mL AlOOH or AlPO4, were measured at 2 min intervals for a total of 20 min. The antigen FHA was selected because it is known to bind to a high extent to both AlOOH and AlPO4 at neutral pH [30]. As expected, the fluorescence intensity of FHA alone showed no changes over time, while a decrease in fluorescence intensity was seen with both aluminum salts (Fig. 2). As the aluminum adjuvants settle, the protein that is bound to the adjuvant gets pulled down below the excitation beam,
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Fig. 2. Fluorescence intensity of antigens alone or adsorbed to adjuvants over time. Fluorescence intensity at 337 nm (280 nm excitation) of 100 mg/mL FHA, and FHA in the presence of 0.66 mg/mL AlOOH and AlPO4, was collected at 2 min intervals, over a period of 20 min (n = 2). The error bars represent the standard deviation from the mean.
Fig. 1. Fluorescence spectra of FHA (A), PRN (B) and FIM (C), at 100 mg/mL in the absence, or presence, of 0.66 mg Al/mL of aluminum salt adjuvant (n = 3).
which caused the observed decrease in fluorescence intensity (Fig. 2). However, the time for which fluorescence intensity was undisturbed varied depending on the adjuvant. Protein adsorbed to AlPO4 was found to settle after about 10 min, while in AlOOH, settling started to occur after about 16 min (Fig. 2). This is likely due to the differences in the sedimentation rate that can be greatly influenced by particle size, shape, density and the magnitude of zeta potential of the aluminum salts. To address the effect of sedimentation, all fluorescence measurements shown in this manuscript were obtained within 2 min of mixing samples in the cuvettes. 3.3. Calibration curves Once detection was proven to be possible in the presence of adjuvants, an investigation of the linear response of fluorescence to protein concentration was conducted. Calibration curves were generated for FHA, PRN and FIM in 0.66 mg Al/mL AlOOH or AlPO4, or in TBS (control), between 1 mg/mL and the highest concentration possible for the protein batch of interest. The slopes of the linear regressions were compared using ANCOVA.
Linear trends can be clearly detected both in the antigen alone and the antigen adsorbed to aluminum salt adjuvants, with correlation coefficients (R2) greater than 0.993 (Fig. 3) (Table 1). For the most part, the addition of adjuvants did result in a slight reduction in the R2 value, specifically for FHA and PRN, which is most likely due to the increased variability brought on by the greater complexity inherent to the adjuvant matrix. Changes in the adjuvant particle size distribution and/or concentration from sample-to-sample in the standard curve would increase variability and have a negative impact on the correlation coefficient. Limit of detection (LOD) and limit of quantification (LOQ) were determined using the ICH guidelines based on the standard deviation of the blank (Table 1) [26]. PRN formulations were found to have, as a whole, the lowest LOD and LOQ, followed by FIM and FHA. This may be related to the higher molar concentration of the fluorophore Trp in PRN compared to FHA. It is estimated that under identical w/v concentration, PRN samples yield about 2.6 times higher molar concentration of Trp than FHA. The addition of AlPO4 to the formulations caused a significant increase (p-value < 0.0001) in the calibration curve slope for all antigens, indicating an increase in sensitivity (Fig. 3). The addition of AlOOH to the formulations also had a statistically significant effect on the calibration curve slopes, though not to the same extent as AlPO4 in all cases. The AlOOH inclusion in the FHA formulation increased the slope (p-value = 0.002), also suggesting an improvement in sensitivity. The presence of AlOOH in PRN and FIM formulation decreased the slope (p-value = 0.0007 and p-val ue < 0.0001, respectively), indicating a drop in sensitivity. Therefore it was established that the calibration curve sensitivity is specific to the antigen-adjuvant combination under consideration. The limit of linearity (LOL) was calculated as the highest concentration of antigen that produced both an R2 0.980 and a p-v alue > 0.05 for the runs test for departure from linearity, and this was found to be antigen dependent (Table 1). The IF assay was acceptably accurate for the formulations tested at nominal concentrations of 50 mg/mL and 100 mg/mL. FIM formulations produced the highest overall percent accuracy of the test samples, followed by FHA, and then PRN (Table 2). In general, lower accuracy was observed with lower concentration of antigens. 3.4. Inter-assay variability Formulations of FHA adsorbed to 0.66 mg Al/mL AlOOH, at low (10 mg/mL), medium (50 mg/mL) and high (100 mg/mL) protein
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Table 2 Accuracy of the intrinsic fluorescence method for quantification of FHA, PRN and FIM. Formulation
Nominal concentration (mg/mL)
Experimentally determined concentrationa (mg/mL)
Percent accuracyb
FHA
10 50 200
8.5 50.0 201.0
85.0 100.0 100.5
FHA-AlOOH
10 50 200
9.9 47.5 184.6
99.0 95.0 92.3
FHA-AlPO4
10 50 200
9.3 53.5 185.8
93.0 107.0 92.9
PRN
10 50 100
7.3 49.3 97.3
73.0 98.6 97.3
PRN-AlOOH
10 50 100
10.3 48.1 97.3
103.0 96.2 97.3
PRN-AlPO4
10 50 100
5.8 44.8 93.8
58.0 89.6 93.8
FIM
10 50 200
9.9 48.8 201.9
99.0 97.6 101.0
FIM-AlOOH
10 50 200
9.8 47.8 196.9
98.0 95.6 98.5
FIM-AlPO4
10 50 200
7.0 47.8 195.0
70.0 95.6 97.5
a Concentrations were calculated by using linear regression of the standard curve. b Percent accuracy = (calculated concentration/nominal concentration) 100.
Table 3 Inter-assay variation of FHA-AlOOH samples. Experimentally determined concentrationa (mg/mL) Fig. 3. Calibration curves of FHA (A), PRN (B), FIM (C). Formulations of the antigen alone and adsorbed to 0.66 mg Al/mL of either AlOOH or AlPO4. Upon excitation at 280 nm, the florescence intensity (FHA at 337 nm, PRN at 337 nm and FIM at 310 nm) was plotted against the concentration (n = 2). The error bars represent the standard deviation from the mean.
Table 1 Coefficient of Determination, LOD and LOQ of Calibration Curves for FHA, PRN and FIM. Standard curve
R2
LOD (mg/mL)
LOQ (mg/mL)
LOL (mg/mL)
FHA FHA-AlOOH FHA-AlPO4
1.000 0.996 0.993
1.4 1.5 1.4
4.3 4.4 4.2
200
PRN PRN-AlOOH PRN-AlPO4
0.999 0.999 0.994
0.2 0.1 0.3
0.6 0.4 1.0
100
FIM FIM-AlOOH FIM-AlPO4
0.999 0.999 0.999
0.3 1.2 0.9
1.0 3.8 2.6
200
concentrations were used to investigate the inter-assay variability. The coefficient of variation (CV) for low, medium and high concentration samples was 7.4%, 1.5%, and 0.6%, respectively, for all 10 experiments performed, indicating high reproducibility of the IF assay (Table 3).
Replicate number
10 mg/mL nominal concentration
50 mg/mL nominal concentration
200 mg/mL nominal concentration
1 2 3 4 5 6 7 8 9 10 Mean SDb CVc (%)
9.1 10.6 9.9 10.6 9.9 10.7 10.3 8.5 10.4 9.3 9.9 0.7 7.4
48.5 50.6 50.1 50.0 48.8 49.2 49.2 50.2 48.5 49.5 49.5 0.8 1.5
196.6 199.8 198.2 198.0 196.7 197.8 198.1 197.1 197.4 200.3 198.0 1.2 0.6
a Replicate concentrations were calculated by using linear regression of the FHAAlOOH standard curve. b Standard deviation. c Coefficient of variation = (standard deviation/mean) 100.
3.5. Effect of adjuvant concentration To investigate the effect of adjuvant concentration, calibration curves for FHA were prepared at low (0.33 mg Al/mL), medium (0.66 mg Al/mL), and high (1.32 mg Al/mL) adjuvant concentrations and compared using ANCOVA. The slopes of the calibration curves changed significantly depending on the concentration of adjuvant, indicating a change
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in sensitivity (Fig. 4). In the case of AlOOH, at 0.33 mg Al/mL, the calibration curve had a significantly lower slope than the 0.66 mg Al/mL and 1.32 mg Al/mL formulations (p-value < 0.0001 for both), though there was no significant difference between the 0.66 mg Al/mL and 1.32 mg Al/mL slopes (p-value = 0.6) (Fig. 4A). For AlPO4, an increase in adjuvant concentration from 0.33 mg Al/mL to 0.66 mg Al/mL significantly increased the slope of the calibration curve (p-value < 0.0001); however, a further increase to 1.32 mg Al/mL produced a decrease in the slope, though still significantly steeper than that of the 0.33 mg Al/mL curve (p-value = 0.01) (Fig. 4B). The R2 value was seen to improve with increasing AlOOH concentration (from 0.989 to 0.999); however, the opposite was obtained for increasing concentrations of AlPO4 (from 0.992 to 0.980). This could be an indication that the increase in AlPO4 content is enhancing the fluorescence closer to the saturation point, perturbing the linearity of the curve. In contrast, the increase in AlOOH concentration may be just enough to provide metal enhanced fluorescence (MEF), while still being below fluorescence saturation. 3.6. Effects of buffers species and pH
Fig. 4. Calibration curves prepared in low, medium, and high adjuvant concentrations. FHA adsorbed to AlOOH (A) and FHA adsorbed to AlPO4 (B) at 0.32 (low), 0.66 (medium), and 1.32 (high) mg Al/mL Al salt (n = 2). The error bars represent the standard deviation from the mean.
The effect of buffer composition and pH was studied by creating test samples of PRN in 0.66 mg Al/mL AlOOH formulated in various buffers species and pHs, and then concentration predictions using a control calibration curve (TBS/pH 7.4) were compared using ANOVA. When different buffer species and pH were compared (Fig. 5A–D) significant differences were observed amongst the test sample groups (p-value = 0.04 and p-value = 0.05, respectively). These results suggest that calibration curves should match the buffer conditions and pH of test samples to ensure accurate
Fig. 5. Effect of buffers and pH on calibration curves and prediction of 100 mg/mL of PRN adsorbed to 0.66 mg/mL AlOOH. PRN adsorbed to 0.66 mg/mL AlOOH calibration curves were made with different buffer compositions and pH. Individual calibration curves for 3 different buffers (TBS, PBS, and HEPES) at a pH of 7.4 (A), and for buffers at 3 different pHs (6, 7.4, and 9) (B) (n = 1). Test samples were made with different buffer compositions and pH and the concentration was predicted using the TBS/pH 7.4 calibration curve. Concentration predictions for 3 different buffers (TBS, PBS, and HEPES) at a pH of 7.4 (C), and for buffers at 3 different pHs (6, 7.4, and 9) (D) (n = 2). The error bars represent the standard deviation from the mean.
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determination of antigen concentrations. Under certain circumstances, such as sample volume limitations or high throughput needs (i.e., buffer composition, pH and ionic strength screening), where accuracy can be leveraged for efficiency or constraints, utilizing a calibration curve of a single buffer condition (or pooling multiple buffer conditions) may be an option.
percent difference across batches for FHA, PRN, FHA-AlPO4 and PRN-AlPO4 was 0.0–16.0%, 2.7–7.1%, 9.0–14.1% and 3.2–6.6%, respectively (Table 5). The results for both adjuvant and nonadjuvanted formulations indicated a very good agreement among micro-Kjeldahl and IF methods (Table 5). 4. Discussion
3.7. Percent adsorption The percent of antigen adsorption is an important parameter that needs to be adjusted to desired levels during formulation development of vaccines [31]. PRN-AlOOH formulations were used as a model to investigate the utility of the IF method to determine the percent adsorption of antigens to aluminum salt adjuvants. To obtain different levels of adsorption, the AlOOH was pretreated with different amounts of phosphate ions. When the concentration of phosphate ions was increased from 0 to 80 mM, a significant increase in the concentration of unbound PRN was detected by IF assay (Table 4), which translated to a decreased percentage of adsorption from 67 to 7.6%. It is important to mention that a decrease in the total antigen concentration was detected in samples treated with increasing concentration of phosphate. These results indicate an impact of either the degree of PRN adsorption or phosphate concentration on the quantification of PRN by IF. 3.8. Comparison with micro-Kjeldahl The IF method was compared against the micro-Kjeldahl technique. This elemental analysis technique determines protein concentration through quantification of organic nitrogen and can be applied to both adjuvanted and non-adjuvanted samples. Samples of unknown antigen concentration (both adjuvanted and unadjuvanted) were measured by micro-Kjeldahl and IF. The
Table 4 Determining percent adsorption using intrinsic fluorescence of 50 mg/mL (nominal) PRN in 0.66 mg/mL AlOOH. Phosphate concentration (mM)
Experimentally determined concentrationa (mg/mL)
Concentration of supernatant (mg/ mL)b
Adsorptionc (%)
0 2 20 80
48.4 42.3 42.6 42.2
15.9 31.3 38.9 39.0
67.2 26.0 8.4 7.6
a Concentrations were determined by using linear regression of the PRN-AlOOH standard curve. b Concentrations were determined by using linear regression of the PRN standard curve. c Fraction adsorbed is the percent of PRN adsorbed to the adjuvant. Fraction adsorbed = (|concentration of total formulation-concentration of supernatant|/concentration of total formulation) 100.
The determination of protein concentration in the presence of aluminum salt adjuvant is an intricate task due to the inherent particulate nature of the adjuvant and the very often strong antigen adsorption to adjuvant particles. To circumvent the turbiditydriven interference, we explored the use of IF for the quantification of proteins in aluminum salt adjuvanted vaccines. Here, we demonstrated that IF from proteins can be detected in the presence of the two more commonly used aluminum salt adjuvants and it can produce linear responses. The results indicated that the IF assay is highly accurate, reproducible and sensitive to quantify protein antigens well within the range of concentrations used in most vaccines. Investigation of potential factors that can influence protein determination by this method indicated that the type of aluminum salt and concentration, type of antigen, buffer specie, and pH, all had an impact on the fluorescence detection. When comparing the effects of the type of aluminum salts, it was found that AlPO4 enhanced the fluorescence intensity of all proteins under evaluation (Fig. 1). This could be attributed to changes in scattering intensity due to alterations in the apparent particle size as protein-coated adjuvant particles agglomerate. The increase in fluorescence intensity in AlPO4 samples could also be due to the MEF phenomenon, which consists of the simultaneous interchange of both enhancements in the emission intensity of the proteins, due to enhanced radiative pathways induced by the aluminum metals, as well as quenching of the protein emission that can occur due to non-radiative energy transmission to the adjuvants [32]. This influence of metallic particles on fluorophores is due to electromagnetic interactions and steady plasmon resonances at the surface, which essentially enhance fluorophore photostability, quantum yield, and energy transfer, and decrease lifetimes [33–37]. Differences in physiochemical characteristics, such as particle size distribution and protein adsorption strength, between AlOOH and AlPO4 [38–40] may be underlying factors causing a stalemate between competing fluorescence enhancement and quenching for AlOOH containing formulations, and an imbalance—favoring fluorescence enhancement, for AlPO4 containing formulations. AlPO4 displayed significant MEF effects, whereas AlOOH produced changes to a much lesser degree, at an identical concentration. Nevertheless, changes in the MEF effect were seen when adjuvant concentrations were increased as both AlOOH and AlPO4 enhanced fluorescence with a doubling in concentration (Fig. 4).
Table 5 Comparison between intrinsic fluorescence and micro-Kjeldahl methods for the determination of FHA and PRN concentration. Antigen
Adjuvant
Lot
Intrinsic fluorescence concentration (mg/mL)a
Micro-Kjeldahl concentration (mg/mL)
Percent differenceb
FHA
None
PRN
None
FHA
0.66 mg Al/mL AlPO4
PRN
0.66 mg Al/mL AlPO4
1 2 1 2 1 2 1 2
1224.8 1143.0 227.7 240.8 1062.7 882.7 158.2 145.3
1225.0 973.4 221.7 224.2 923.0 807.0 148.0 150.0
0.0 16.0 2.7 7.1 14.1 9.0 6.6 3.2
a
Concentrations were determined by using linear regression of the respective intrinsic fluorescence standard curve. Percent difference = [(calculated concentration from technique 1 calculated concentration from technique 2)/(|calculated concentration from technique 1 + calculated concentration from technique 2|)/2] 100. b
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The type of protein was also found to influence linearity, slope of calibration curves, and therefore, LOD and LOQ. This is likely related to the relative molar concentration of aromatic amino acids Trp and Tyr, which are responsible for most of the fluorescence emission in proteins when excited at 280 nm. In addition, the location of the relevant aromatic amino acids may also have also an impact on the fluorescence intensity as collisional quenching by solution ions has significantly more effect on surface fluorophores compared to their buried counterparts [24]. Changes in buffer and pH were found to affect calibration curve predictions significantly. This could be due to buffer/pH induced changes in antigens’ tertiary structure, impacting the exposure of the aromatic amino acids, ultimately affecting the fluorescence intensities. Therefore it is important to factor in formulation characteristics like adjuvant type, adjuvant concentration, buffer, and pH, when establishing standard curves for the IF assay. Since the method is based on detection of intrinsic fluorescence, one of the main disadvantages is the inability to detect proteins or peptides lacking of aromatic amino acid residues. Nevertheless, many advantages are apparent for the IF method when compared to traditional methods normally used for the determination of protein concentration in vaccines. In contrast to classic methods, the IF assay is extremely simple, non-destructive, and requires minimal assay development. In this context, the IF method was shown to produce comparable results with micro-Kjeldahl, a method that is widely used for the determination of total protein in biopharmaceutical products and vaccines [13,41]. Another advantage of the IF method is that it can be directly applied to the vaccine sample without the need for desorption, dissolution or chemical reactions. The nondestructive and non-invasive nature of IF are maybe the most important features of the assay, which are essential for the implementation of Process Analytical Technology (PAT) tools and to support Quality by Design (QbD) approaches [42,43]. The PAT concept actually aims at understanding the processes by monitoring quality attributes in real time [43]. As such, there is a need for non-destructive methods of quantification that can be applied in/ on-line to increase testing efficiency and consistency, while also reducing over-processing and number of rejects. It is highly conceivable that the IF method can be adapted for in-process monitoring of antigen concentration during vaccine manufacturing using in-line fluorimeters [44]. In conclusion, we have described a direct, non-destructive, simple and inexpensive fluorimetric assay for the determination of protein concentration in vaccines containing commonly used aluminum salt adjuvants. The method exhibited high accuracy, sensitivity and reproducibility within the protein concentration range found in most vaccines. The application of this technique is not limited to the determination of protein concentration in aluminum adjuvant containing vaccines, as it has great potential for the quantification of proteins formulated with other types of adjuvant systems, such as cationic peptide-CpG, squalene emulsions, saponins, liposomes, etc. Studies looking at the applicability of the IF method to these adjuvant systems are underway. Acknowledgements The authors would like to express their cordial thanks to Michelle Nieuwesteeg for technical assistance in the preparation of this manuscript. Disclosure of potential conflict of interest During the period in which this work was performed all authors of this manuscript were Sanofi Pasteur’s employees.
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Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
An intrinsic fluorescence method for the determination of protein concentration in vaccines containing aluminum salt adjuvants Lucienne Nouchikian 1, Cristopher Roque, Jimmy Y. Song 2, Nausheen Rahman, Salvador F. Ausar ⇑ BioProcess Research and Development, Sanofi Pasteur, 1755 Steeles Ave West, Toronto, Ontario M3R 3T4, Canada
a r t i c l e
i n f o
Article history: Received 20 April 2018 Received in revised form 12 July 2018 Accepted 3 August 2018 Available online xxxx Keywords: Intrinsic fluorescence Adjuvants Aluminum salt adjuvant Vaccines Protein quantification
a b s t r a c t Determination of protein concentration in vaccines containing aluminum salt adjuvant typically necessitates desorption of the protein prior to analysis. Here we describe a method based on the intrinsic fluorescence of tyrosine and tryptophan that requires no desorption of proteins. Adjuvanted formulations of three model Bordetella pertussis antigens were excited at 280 nm and their emission spectra collected from 290 to 400 nm. Emission spectra of protein antigens in the presence of aluminum salt adjuvants were able to be detected, the effects of adjuvants on the spectra were analyzed, and linear regressions were calculated. The fluorescence method proved to be very sensitive with a limit of quantification between 0.4 and 4.4 mg/mL and limit of linearity between 100 and 200 mg/mL, across the formulations tested. The fluorescence method was found to be influenced by adjuvant presence, type of adjuvant, adjuvant concentration, buffer and pH conditions. The method also demonstrated ability to monitor the percent adsorption of antigens to the adjuvants. Furthermore, intrinsic fluorescence showed good correlation with micro-Kjeldahl elemental assay in quantifying protein concentration. Being a non-invasive, quick and sensitive method, intrinsic fluorescence has the potential to be utilized as a high throughput tool for vaccine development and conceivably implemented in-line, using in-line fluorimeters, to monitor antigen concentration during formulation processing. Ó 2018 Published by Elsevier Ltd.
1. Introduction The concentration of antigens in vaccine products is an important parameter that needs to be controlled to ensure consistency in the manufacturing process and product quality. Monitoring the concentration of antigens during processing is important for several reasons such as controlling formulation accuracy, estimating process-related losses, monitoring product homogeneity, and ensuring appropriate adsorption of antigens to a given adjuvant [1,2]. Typically protein-based vaccines are presented as suspensions or emulsions due to the presence of adjuvants such as aluminum salts (i.e., AlOOH, AlPO4, etc.), or squalene emulsions (i.e., squalene AS03 emulsion, MF59, etc.). Adjuvants are a key component in vaccines [3–5]; however, the presence of adjuvants can produce significant interference with commonly used methods for protein ⇑ Corresponding author. E-mail address: [email protected] (S.F. Ausar). Present address: Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada. 2 Present address: Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66047, USA. 1
quantification, due to turbidity introduced by the adjuvant particles. For the case of vaccines containing aluminum salts, many methods have been published for analysis of antigens, but they necessitate antigen desorption prior to testing due to the turbidity of the formulations [6–8]. Desorption procedures described in the literature are rather complex and time-consuming, with variable recovery and questionable reproducibility [9,10]. Other methods reported for the quantification of antigens in adjuvanted vaccines, such as, flow cytometry [11], chemiluminescent nitrogen detection [12], micro-kjeldalh [13] and o-phthalaldehyde assay [9], can be directly applied to suspensions. However, these methods are mostly destructive, requiring specific antibodies or special reagents, and are therefore not suitable for fast turnaround or in-line process monitoring. Proteins are unique among biomolecules in displaying intrinsic fluorescence (IF) due to the presence of aromatic amino acids. Most proteins contain aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe). Upon excitation in the UV region (250 nm for Phe, 275 nm Tyr and 290 nm for Trp), these aromatic amino acids can produce an emission spectrum with maximum emission in the range of 310–360 nm [14], in which the intensity is proportional to the protein concentration.
https://doi.org/10.1016/j.vaccine.2018.08.005 0264-410X/Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Nouchikian L et al. An intrinsic fluorescence method for the determination of protein concentration in vaccines containing aluminum salt adjuvants. Vaccine (2018), https://doi.org/10.1016/j.vaccine.2018.08.005
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Amongst the three fluorescent amino acids found in proteins, Phe contribution to the IF of protein is negligible due to the low quantum yield. Although Trp and Tyr possess similar quantum yields, the indole group of Trp has been reported to be the dominant source of UV absorbance near 280 nm and fluorescence emission near 330 nm in proteins [15]. Despite the relatively inferior fluorescence properties of Tyr, analysis of its IF has been shown to be a useful tool for analysis of proteins that are lacking Trp [16]. Many reports in the literature have described the use of IF, especially the use of Trp, to study changes in emission wavelength and intensity, in order to analyze conformational changes in experimental vaccines containing toxoids [17], inactivated [18] and attenuated viruses [19], virus like particles [20], and aluminum salt adsorbed proteins [21,22]. However, the use of IF to monitor protein concentration has not been well explored. Previous research from our laboratory has indicated that it is possible to detect the fluorescence of extrinsically added dyes to formulations containing aluminum salt adjuvants with no detectable adjuvant interference [23]. Based on that premise, we hypothesize that the aluminum salts would not significantly interfere with the IF of proteins, and it could serve as a method for protein quantification in a matrix of mineral salt adjuvants. This is due to the fact that fluorescence is collected at a 90° angle, which reduces the inner filtering effects from sample turbidity [24]. In this paper we use the Bordetella pertussis antigens Filamentous haemagglutinin (FHA), Pertactin (PRN) and Fimbria (FIM), to demonstrate that IF of proteins can be detected in formulations containing aluminum salts allowing the development of a rapid, simple and non-destructive method of quantifying antigen concentration in vaccine formulations. The method was developed for antigens formulated in aluminum phosphate (AlPO4) and aluminum oxyhydroxide (AlOOH), as these are the most commonly used aluminum salts in vaccines.
then mixed on an AdamsTM Nutator Mixer (Becton Dickinson, New Jersey, United States) at room temperature for 30 min to allow for antigen adsorption to the adjuvant. Preliminary experiments indicated that the percentage adsorption does not change significantly after 30 minutes (min) of mixing with the adjuvant. Unless otherwise stated, the buffer used for the standard curves and test samples was TBS. However, other buffers (PBS, HEPES, citrate, and bicarbonate) were used to test the effect of buffer and pH conditions on the calibration curves. To test the effect of percent adsorption, different concentrations of phosphate (0 mM, 2 mM, 20 mM, and 80 mM) were added to the formulations in TBS. Test samples containing FHA, prepared to assess the effect of adjuvant presence and inter-assay variation on calibration curve accuracy, were made at antigen concentrations of 10, 50 and 200 mg/mL, with an adjuvant concentration of 0.66 mg Al/mL for the formulations containing either AlOOH or AlPO4. Test samples containing FHA, created to assess the effect of sedimentation, were prepared at an antigen concentration of 100 mg/mL, with 0.66 mg Al/mL adjuvant concentration for the formulations containing either AlOOH or AlPO4. Test samples containing PRN, created to assess the effect of adjuvant presence on calibration curve accuracy, were prepared at antigen concentrations of 10, 50 and 100 mg/mL, with an adjuvant concentration of 0.66 mg Al/mL for the formulations containing either AlOOH or AlPO4. Test samples containing PRN, created to assess the effect of buffer and pH on calibration curve prediction accuracy, were prepared at an antigen concentration of 100 mg/mL, with an adjuvant concentration of 0.66 mg Al/mL for the formulations containing either AlOOH or AlPO4. Test samples containing FIM, prepared to assess the effect of adjuvant presence on calibration curve accuracy, were prepared at antigen concentrations of 10, 50 and 200 mg/mL, with an adjuvant concentration of 0.66 mg Al/mL for the formulations containing either AlOOH or AlPO4.
2. Materials and methods 2.3. Fluorescence measurements 2.1. Materials Filamentous haemagglutinin (FHA), molecular weight (MW) = 220 kDa, pI = 8.70; Pertactin (PRN), MW = 60.3 kDa, pI = 6.03; Fimbriae (FIM), type 2, MW = 19.2 kDa; pI = 5.94, and type 3, MW = 17.2 kDa, pI = 9.13, were manufactured and provided by Sanofi Pasteur (Toronto, Canada). With respect to aromatic amino acids residues relevant to IF, it is important to mention that FHA has 11 tryptophan and 28 tyrosine, PRN has 8 tryptophan and 6 tyrosine, FIM Type 2 has no tryptophan and 8 tyrosine, and FIM Type 3 has no tryptophan and 7 tyrosine. Batches of FHA, PRN and FIM were prepared at concentrations ranging from 973.4–1200.9 mg/mL, 147.7–241.9 mg/mL and 242. 0–402.0 mg/mL, respectively. The AlOOH adjuvant was supplied at 10.2 mg Al/mL by Brenntag Biosector (Frederikssund, Denmark). The AlPO4 adjuvant was prepared by Sanofi Pasteur (Toronto, Canada) at 4.1 mg Al/mL. The buffers 10 mM Tris-HCl with 150 mM NaCl, pH 7.4 (TBS); phosphate buffered saline (PBS); 150 mM citrate, pH 6; 100 mM bicarbonate, pH 9; and 0.5 M phosphate, pH 7.4; were made in house with reagents supplied by Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). HEPES was supplied by Life Technologies at 1 M concentrated solution and diluted to 10 mM with Milli-Q water and the pH was adjusted using 1 M HCl. 2.2. Preparation of formulations All formulations were prepared by adding the appropriate amount of adjuvant to the applicable amount of protein, and then using the designated buffer as a final diluent. Formulations were
Fluorescence measurements of all formulations were collected using Chirascan Plus spectrophotometer (Applied Photophysics, Surrey, United Kingdom). The samples were excited at an excitation wavelength of 280 nm and the absolute fluorescence intensities were collected between 290 and 400 nm, at 1 nm increments and a measurement time per point of 0.5 s, using a constant photomultiplier tube setting of 800 V and a set temperature of 20 °C. Once a maximum emission peak was found, the spectra collection was set to ±20 nm from the maximum emission peak. Standard 10 mm square quartz cuvettes were used to measure the formulations. The use of square geometry cuvettes provided better spectral quality when compared to the triangular front-face counterpart (data not shown). Readings were taken immediately after mixing for optimal results. 2.4. Sedimentation study FHA samples were mixed by 10 repeated inversions, and the fluorescence intensity from 290 to 400 nm was collected at 2 min intervals, for 20 min. The fluorescence intensity, at 337 nm, was averaged between the duplicates. 2.5. Calibration curves Calibration curves were created in duplicate following International Standard ISO 11095 [25]. The FHA calibration curves created to assess the effect of adjuvant were made with samples at antigen concentrations of 0, 1, 5, 10, 50, 100 and 200 mg/mL, and an adjuvant concentration of 0.66 mg Al/mL for the formulations
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containing either AlOOH or AlPO4. The FHA calibration curves created to assess the effect of adjuvant concentration were made with samples at antigen concentrations of 5, 10, 50, 100 and 200 mg/mL, and AlOOH or AlPO4 concentrations of 0.33 mg Al/mL, 0.66 mg Al/ mL and 1.32 mg Al/mL. The PRN calibration curves created to assess the effect of adjuvant were made with samples at antigen concentrations of 0, 1, 5, 10, 50 and 100 mg/mL, and an adjuvant concentration of 0.66 mg Al/mL for the formulations containing either AlOOH or AlPO4. The PRN calibration curves created to assess the effect of buffer composition and pH, were made with samples at antigen concentrations of 0, 5, 10, 50 and 100 mg/mL, and an AlOOH concentration of 0.66 mg Al/mL. The FIM calibration curves created to assess the effect of adjuvant were made with samples at antigen concentrations of 0, 1, 5, 10, 50, 100, 150 and 200 mg/mL, and an adjuvant concentration of 0.66 mg Al/mL for the formulations containing either AlOOH or AlPO4. The wavelength at which the maximum intensity was reached was selected and the fluorescence at this point was averaged between duplicates and plotted against its concentration. For the calibration curves generated to assess the effect of adjuvant and adjuvant concentration, the fluorescence intensities were averaged between the duplicates. For all other calibration curves, no replicates were obtained. Limit of detection (LOD) and limit of quantification (LOQ) were calculated using the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guide lines [26], as follows: LOD = 3.3r/S LOQ = 10r/S r = standard deviation of the blank S = slope of the linear regression curve. Limit of linearity (LOL) was established by removing data points, starting from the highest concentration of antigen, from the calibration curve, until both the R2 of the linear regression was 0.980 and the runs test for departure from linearity produced a p-value > 0.05, using GraphPad Prism version 6.00 (GraphPad Software, La Jolla California, USA). Statistical comparison of test samples and linear regression slopes were performed by analysis of variance (ANOVA) and analysis of covariance (ANCOVA), respectively, using GraphPad Prism version 7.02 (GraphPad Software, La Jolla California, USA). Test samples and slopes were considered to be statistically different if the probability (p-value) of obtaining a test statistic, F, greater than the one calculated by pure chance was valued at 0.05 or less. 2.6. Inter-assay variability Inter-assay variability was investigated by measuring 10 aliquots of test samples, prepared at low (10 mg/mL), medium (50 mg/mL) and high (200 mg/mL) concentrations of FHA adsorbed to 0.66 mg Al/mL AlOOH, against 10 aliquoted calibration curves of FHA-AlOOH formulations. 2.7. Percent adsorption Percent adsorption was tested with 50 mg/mL of PRN adsorbed to 0.66 mg Al/mL AlOOH with the presence of 0 mM, 2 mM, 20 mM and 80 mM phosphate, to create a difference in the percent adsorption levels. The fluorescence of the total formulations was collected, along with the fluorescence of the supernatant, after centrifugation at 20,000 x g for 10 min on an Eppendorf 5417 centrifuge (Eppendorf, Germany). The concentration of the
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formulations, and the supernatant, was back-calculated using their respective standard curves. 2.8. Comparison with micro-Kjeldahl Samples of unknown antigen concentrations were measured by both micro-Kjeldahl [27] and IF methods. For the IF method, adjuvant free samples were diluted in TBS, and samples containing AlPO4 were diluted in TBS containing 0.66 mg Al/mL of AlPO4, until a concentration value within the respective calibration curve range was obtained. All dilutions were back calculated using the linear regression of their respective standard curve. The back calculated concentrations were compared against the micro-Kjeldahl results. 3. Results 3.1. Effect of aluminum salt adjuvants on the fluorescence spectrum of protein antigens To investigate the feasibility of the IF method for the detection of protein concentration in the presence of aluminum salt adjuvants, formulations of FHA, PRN and FIM, at 100 mg/mL, in the presence of AlOOH or AlPO4, were excited at 280 nm and their full emission spectra from 290 to 400 nm was immediately collected in square 10 mm cuvettes (Fig. 1). In the absence of aluminum salts, both FHA and PRN showed broad and well detectable peaks, with a maximum at 337 nm ± 1 nm (n = 3), (Fig. 1A and B), while FIM displayed a narrower peak with maximum fluorescence intensity at 310 nm ± 0 nm (n = 3) (Fig. 1C). The lower emission wavelength detected in FIM is attributed to a lack of tryptophan in its structure, making tyrosine the primary contributor to the fluorescence spectrum [28]. The addition of aluminum salts did not significantly affect the maximum emission wavelength. However, the fluorescence intensity was impacted depending on the type of adjuvant used in the formulation. While a significant increase in the fluorescence intensity was observed for AlPO4 containing samples, the presence of AlOOH did not notably impact the fluorescence spectra of the proteins under investigation (Fig. 1). To investigate any potential time-dependent structural changes that might influence the fluorescence signals during data acquisition, the peak position of the Trp fluorescence of FHA as a function of time was analyzed. No noticeable changes were observed on the peak position of the fluorescence spectra of FHA for up to 20 min of incubation at room temperature (Supplementary Fig. 1). Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.vaccine.2018.08. 005. Altogether, the results indicate that the presence of aluminum salts at adjuvant concentrations commonly used in vaccines does not interfere with the fluorescence properties of proteins, making this method feasible for the detection of proteins in aluminum salt adjuvanted vaccines. 3.2. Effect of sedimentation Suspensions of aluminum salt adjuvants are known to sediment over time [29]. To investigate the effect of sedimentation on the IF, the fluorescence intensity of formulations containing 100 mg/mL FHA alone, or adsorbed to 0.66 mg Al/mL AlOOH or AlPO4, were measured at 2 min intervals for a total of 20 min. The antigen FHA was selected because it is known to bind to a high extent to both AlOOH and AlPO4 at neutral pH [30]. As expected, the fluorescence intensity of FHA alone showed no changes over time, while a decrease in fluorescence intensity was seen with both aluminum salts (Fig. 2). As the aluminum adjuvants settle, the protein that is bound to the adjuvant gets pulled down below the excitation beam,
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Fig. 2. Fluorescence intensity of antigens alone or adsorbed to adjuvants over time. Fluorescence intensity at 337 nm (280 nm excitation) of 100 mg/mL FHA, and FHA in the presence of 0.66 mg/mL AlOOH and AlPO4, was collected at 2 min intervals, over a period of 20 min (n = 2). The error bars represent the standard deviation from the mean.
Fig. 1. Fluorescence spectra of FHA (A), PRN (B) and FIM (C), at 100 mg/mL in the absence, or presence, of 0.66 mg Al/mL of aluminum salt adjuvant (n = 3).
which caused the observed decrease in fluorescence intensity (Fig. 2). However, the time for which fluorescence intensity was undisturbed varied depending on the adjuvant. Protein adsorbed to AlPO4 was found to settle after about 10 min, while in AlOOH, settling started to occur after about 16 min (Fig. 2). This is likely due to the differences in the sedimentation rate that can be greatly influenced by particle size, shape, density and the magnitude of zeta potential of the aluminum salts. To address the effect of sedimentation, all fluorescence measurements shown in this manuscript were obtained within 2 min of mixing samples in the cuvettes. 3.3. Calibration curves Once detection was proven to be possible in the presence of adjuvants, an investigation of the linear response of fluorescence to protein concentration was conducted. Calibration curves were generated for FHA, PRN and FIM in 0.66 mg Al/mL AlOOH or AlPO4, or in TBS (control), between 1 mg/mL and the highest concentration possible for the protein batch of interest. The slopes of the linear regressions were compared using ANCOVA.
Linear trends can be clearly detected both in the antigen alone and the antigen adsorbed to aluminum salt adjuvants, with correlation coefficients (R2) greater than 0.993 (Fig. 3) (Table 1). For the most part, the addition of adjuvants did result in a slight reduction in the R2 value, specifically for FHA and PRN, which is most likely due to the increased variability brought on by the greater complexity inherent to the adjuvant matrix. Changes in the adjuvant particle size distribution and/or concentration from sample-to-sample in the standard curve would increase variability and have a negative impact on the correlation coefficient. Limit of detection (LOD) and limit of quantification (LOQ) were determined using the ICH guidelines based on the standard deviation of the blank (Table 1) [26]. PRN formulations were found to have, as a whole, the lowest LOD and LOQ, followed by FIM and FHA. This may be related to the higher molar concentration of the fluorophore Trp in PRN compared to FHA. It is estimated that under identical w/v concentration, PRN samples yield about 2.6 times higher molar concentration of Trp than FHA. The addition of AlPO4 to the formulations caused a significant increase (p-value < 0.0001) in the calibration curve slope for all antigens, indicating an increase in sensitivity (Fig. 3). The addition of AlOOH to the formulations also had a statistically significant effect on the calibration curve slopes, though not to the same extent as AlPO4 in all cases. The AlOOH inclusion in the FHA formulation increased the slope (p-value = 0.002), also suggesting an improvement in sensitivity. The presence of AlOOH in PRN and FIM formulation decreased the slope (p-value = 0.0007 and p-val ue < 0.0001, respectively), indicating a drop in sensitivity. Therefore it was established that the calibration curve sensitivity is specific to the antigen-adjuvant combination under consideration. The limit of linearity (LOL) was calculated as the highest concentration of antigen that produced both an R2 0.980 and a p-v alue > 0.05 for the runs test for departure from linearity, and this was found to be antigen dependent (Table 1). The IF assay was acceptably accurate for the formulations tested at nominal concentrations of 50 mg/mL and 100 mg/mL. FIM formulations produced the highest overall percent accuracy of the test samples, followed by FHA, and then PRN (Table 2). In general, lower accuracy was observed with lower concentration of antigens. 3.4. Inter-assay variability Formulations of FHA adsorbed to 0.66 mg Al/mL AlOOH, at low (10 mg/mL), medium (50 mg/mL) and high (100 mg/mL) protein
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Table 2 Accuracy of the intrinsic fluorescence method for quantification of FHA, PRN and FIM. Formulation
Nominal concentration (mg/mL)
Experimentally determined concentrationa (mg/mL)
Percent accuracyb
FHA
10 50 200
8.5 50.0 201.0
85.0 100.0 100.5
FHA-AlOOH
10 50 200
9.9 47.5 184.6
99.0 95.0 92.3
FHA-AlPO4
10 50 200
9.3 53.5 185.8
93.0 107.0 92.9
PRN
10 50 100
7.3 49.3 97.3
73.0 98.6 97.3
PRN-AlOOH
10 50 100
10.3 48.1 97.3
103.0 96.2 97.3
PRN-AlPO4
10 50 100
5.8 44.8 93.8
58.0 89.6 93.8
FIM
10 50 200
9.9 48.8 201.9
99.0 97.6 101.0
FIM-AlOOH
10 50 200
9.8 47.8 196.9
98.0 95.6 98.5
FIM-AlPO4
10 50 200
7.0 47.8 195.0
70.0 95.6 97.5
a Concentrations were calculated by using linear regression of the standard curve. b Percent accuracy = (calculated concentration/nominal concentration) 100.
Table 3 Inter-assay variation of FHA-AlOOH samples. Experimentally determined concentrationa (mg/mL) Fig. 3. Calibration curves of FHA (A), PRN (B), FIM (C). Formulations of the antigen alone and adsorbed to 0.66 mg Al/mL of either AlOOH or AlPO4. Upon excitation at 280 nm, the florescence intensity (FHA at 337 nm, PRN at 337 nm and FIM at 310 nm) was plotted against the concentration (n = 2). The error bars represent the standard deviation from the mean.
Table 1 Coefficient of Determination, LOD and LOQ of Calibration Curves for FHA, PRN and FIM. Standard curve
R2
LOD (mg/mL)
LOQ (mg/mL)
LOL (mg/mL)
FHA FHA-AlOOH FHA-AlPO4
1.000 0.996 0.993
1.4 1.5 1.4
4.3 4.4 4.2
200
PRN PRN-AlOOH PRN-AlPO4
0.999 0.999 0.994
0.2 0.1 0.3
0.6 0.4 1.0
100
FIM FIM-AlOOH FIM-AlPO4
0.999 0.999 0.999
0.3 1.2 0.9
1.0 3.8 2.6
200
concentrations were used to investigate the inter-assay variability. The coefficient of variation (CV) for low, medium and high concentration samples was 7.4%, 1.5%, and 0.6%, respectively, for all 10 experiments performed, indicating high reproducibility of the IF assay (Table 3).
Replicate number
10 mg/mL nominal concentration
50 mg/mL nominal concentration
200 mg/mL nominal concentration
1 2 3 4 5 6 7 8 9 10 Mean SDb CVc (%)
9.1 10.6 9.9 10.6 9.9 10.7 10.3 8.5 10.4 9.3 9.9 0.7 7.4
48.5 50.6 50.1 50.0 48.8 49.2 49.2 50.2 48.5 49.5 49.5 0.8 1.5
196.6 199.8 198.2 198.0 196.7 197.8 198.1 197.1 197.4 200.3 198.0 1.2 0.6
a Replicate concentrations were calculated by using linear regression of the FHAAlOOH standard curve. b Standard deviation. c Coefficient of variation = (standard deviation/mean) 100.
3.5. Effect of adjuvant concentration To investigate the effect of adjuvant concentration, calibration curves for FHA were prepared at low (0.33 mg Al/mL), medium (0.66 mg Al/mL), and high (1.32 mg Al/mL) adjuvant concentrations and compared using ANCOVA. The slopes of the calibration curves changed significantly depending on the concentration of adjuvant, indicating a change
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in sensitivity (Fig. 4). In the case of AlOOH, at 0.33 mg Al/mL, the calibration curve had a significantly lower slope than the 0.66 mg Al/mL and 1.32 mg Al/mL formulations (p-value < 0.0001 for both), though there was no significant difference between the 0.66 mg Al/mL and 1.32 mg Al/mL slopes (p-value = 0.6) (Fig. 4A). For AlPO4, an increase in adjuvant concentration from 0.33 mg Al/mL to 0.66 mg Al/mL significantly increased the slope of the calibration curve (p-value < 0.0001); however, a further increase to 1.32 mg Al/mL produced a decrease in the slope, though still significantly steeper than that of the 0.33 mg Al/mL curve (p-value = 0.01) (Fig. 4B). The R2 value was seen to improve with increasing AlOOH concentration (from 0.989 to 0.999); however, the opposite was obtained for increasing concentrations of AlPO4 (from 0.992 to 0.980). This could be an indication that the increase in AlPO4 content is enhancing the fluorescence closer to the saturation point, perturbing the linearity of the curve. In contrast, the increase in AlOOH concentration may be just enough to provide metal enhanced fluorescence (MEF), while still being below fluorescence saturation. 3.6. Effects of buffers species and pH
Fig. 4. Calibration curves prepared in low, medium, and high adjuvant concentrations. FHA adsorbed to AlOOH (A) and FHA adsorbed to AlPO4 (B) at 0.32 (low), 0.66 (medium), and 1.32 (high) mg Al/mL Al salt (n = 2). The error bars represent the standard deviation from the mean.
The effect of buffer composition and pH was studied by creating test samples of PRN in 0.66 mg Al/mL AlOOH formulated in various buffers species and pHs, and then concentration predictions using a control calibration curve (TBS/pH 7.4) were compared using ANOVA. When different buffer species and pH were compared (Fig. 5A–D) significant differences were observed amongst the test sample groups (p-value = 0.04 and p-value = 0.05, respectively). These results suggest that calibration curves should match the buffer conditions and pH of test samples to ensure accurate
Fig. 5. Effect of buffers and pH on calibration curves and prediction of 100 mg/mL of PRN adsorbed to 0.66 mg/mL AlOOH. PRN adsorbed to 0.66 mg/mL AlOOH calibration curves were made with different buffer compositions and pH. Individual calibration curves for 3 different buffers (TBS, PBS, and HEPES) at a pH of 7.4 (A), and for buffers at 3 different pHs (6, 7.4, and 9) (B) (n = 1). Test samples were made with different buffer compositions and pH and the concentration was predicted using the TBS/pH 7.4 calibration curve. Concentration predictions for 3 different buffers (TBS, PBS, and HEPES) at a pH of 7.4 (C), and for buffers at 3 different pHs (6, 7.4, and 9) (D) (n = 2). The error bars represent the standard deviation from the mean.
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determination of antigen concentrations. Under certain circumstances, such as sample volume limitations or high throughput needs (i.e., buffer composition, pH and ionic strength screening), where accuracy can be leveraged for efficiency or constraints, utilizing a calibration curve of a single buffer condition (or pooling multiple buffer conditions) may be an option.
percent difference across batches for FHA, PRN, FHA-AlPO4 and PRN-AlPO4 was 0.0–16.0%, 2.7–7.1%, 9.0–14.1% and 3.2–6.6%, respectively (Table 5). The results for both adjuvant and nonadjuvanted formulations indicated a very good agreement among micro-Kjeldahl and IF methods (Table 5). 4. Discussion
3.7. Percent adsorption The percent of antigen adsorption is an important parameter that needs to be adjusted to desired levels during formulation development of vaccines [31]. PRN-AlOOH formulations were used as a model to investigate the utility of the IF method to determine the percent adsorption of antigens to aluminum salt adjuvants. To obtain different levels of adsorption, the AlOOH was pretreated with different amounts of phosphate ions. When the concentration of phosphate ions was increased from 0 to 80 mM, a significant increase in the concentration of unbound PRN was detected by IF assay (Table 4), which translated to a decreased percentage of adsorption from 67 to 7.6%. It is important to mention that a decrease in the total antigen concentration was detected in samples treated with increasing concentration of phosphate. These results indicate an impact of either the degree of PRN adsorption or phosphate concentration on the quantification of PRN by IF. 3.8. Comparison with micro-Kjeldahl The IF method was compared against the micro-Kjeldahl technique. This elemental analysis technique determines protein concentration through quantification of organic nitrogen and can be applied to both adjuvanted and non-adjuvanted samples. Samples of unknown antigen concentration (both adjuvanted and unadjuvanted) were measured by micro-Kjeldahl and IF. The
Table 4 Determining percent adsorption using intrinsic fluorescence of 50 mg/mL (nominal) PRN in 0.66 mg/mL AlOOH. Phosphate concentration (mM)
Experimentally determined concentrationa (mg/mL)
Concentration of supernatant (mg/ mL)b
Adsorptionc (%)
0 2 20 80
48.4 42.3 42.6 42.2
15.9 31.3 38.9 39.0
67.2 26.0 8.4 7.6
a Concentrations were determined by using linear regression of the PRN-AlOOH standard curve. b Concentrations were determined by using linear regression of the PRN standard curve. c Fraction adsorbed is the percent of PRN adsorbed to the adjuvant. Fraction adsorbed = (|concentration of total formulation-concentration of supernatant|/concentration of total formulation) 100.
The determination of protein concentration in the presence of aluminum salt adjuvant is an intricate task due to the inherent particulate nature of the adjuvant and the very often strong antigen adsorption to adjuvant particles. To circumvent the turbiditydriven interference, we explored the use of IF for the quantification of proteins in aluminum salt adjuvanted vaccines. Here, we demonstrated that IF from proteins can be detected in the presence of the two more commonly used aluminum salt adjuvants and it can produce linear responses. The results indicated that the IF assay is highly accurate, reproducible and sensitive to quantify protein antigens well within the range of concentrations used in most vaccines. Investigation of potential factors that can influence protein determination by this method indicated that the type of aluminum salt and concentration, type of antigen, buffer specie, and pH, all had an impact on the fluorescence detection. When comparing the effects of the type of aluminum salts, it was found that AlPO4 enhanced the fluorescence intensity of all proteins under evaluation (Fig. 1). This could be attributed to changes in scattering intensity due to alterations in the apparent particle size as protein-coated adjuvant particles agglomerate. The increase in fluorescence intensity in AlPO4 samples could also be due to the MEF phenomenon, which consists of the simultaneous interchange of both enhancements in the emission intensity of the proteins, due to enhanced radiative pathways induced by the aluminum metals, as well as quenching of the protein emission that can occur due to non-radiative energy transmission to the adjuvants [32]. This influence of metallic particles on fluorophores is due to electromagnetic interactions and steady plasmon resonances at the surface, which essentially enhance fluorophore photostability, quantum yield, and energy transfer, and decrease lifetimes [33–37]. Differences in physiochemical characteristics, such as particle size distribution and protein adsorption strength, between AlOOH and AlPO4 [38–40] may be underlying factors causing a stalemate between competing fluorescence enhancement and quenching for AlOOH containing formulations, and an imbalance—favoring fluorescence enhancement, for AlPO4 containing formulations. AlPO4 displayed significant MEF effects, whereas AlOOH produced changes to a much lesser degree, at an identical concentration. Nevertheless, changes in the MEF effect were seen when adjuvant concentrations were increased as both AlOOH and AlPO4 enhanced fluorescence with a doubling in concentration (Fig. 4).
Table 5 Comparison between intrinsic fluorescence and micro-Kjeldahl methods for the determination of FHA and PRN concentration. Antigen
Adjuvant
Lot
Intrinsic fluorescence concentration (mg/mL)a
Micro-Kjeldahl concentration (mg/mL)
Percent differenceb
FHA
None
PRN
None
FHA
0.66 mg Al/mL AlPO4
PRN
0.66 mg Al/mL AlPO4
1 2 1 2 1 2 1 2
1224.8 1143.0 227.7 240.8 1062.7 882.7 158.2 145.3
1225.0 973.4 221.7 224.2 923.0 807.0 148.0 150.0
0.0 16.0 2.7 7.1 14.1 9.0 6.6 3.2
a
Concentrations were determined by using linear regression of the respective intrinsic fluorescence standard curve. Percent difference = [(calculated concentration from technique 1 calculated concentration from technique 2)/(|calculated concentration from technique 1 + calculated concentration from technique 2|)/2] 100. b
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The type of protein was also found to influence linearity, slope of calibration curves, and therefore, LOD and LOQ. This is likely related to the relative molar concentration of aromatic amino acids Trp and Tyr, which are responsible for most of the fluorescence emission in proteins when excited at 280 nm. In addition, the location of the relevant aromatic amino acids may also have also an impact on the fluorescence intensity as collisional quenching by solution ions has significantly more effect on surface fluorophores compared to their buried counterparts [24]. Changes in buffer and pH were found to affect calibration curve predictions significantly. This could be due to buffer/pH induced changes in antigens’ tertiary structure, impacting the exposure of the aromatic amino acids, ultimately affecting the fluorescence intensities. Therefore it is important to factor in formulation characteristics like adjuvant type, adjuvant concentration, buffer, and pH, when establishing standard curves for the IF assay. Since the method is based on detection of intrinsic fluorescence, one of the main disadvantages is the inability to detect proteins or peptides lacking of aromatic amino acid residues. Nevertheless, many advantages are apparent for the IF method when compared to traditional methods normally used for the determination of protein concentration in vaccines. In contrast to classic methods, the IF assay is extremely simple, non-destructive, and requires minimal assay development. In this context, the IF method was shown to produce comparable results with micro-Kjeldahl, a method that is widely used for the determination of total protein in biopharmaceutical products and vaccines [13,41]. Another advantage of the IF method is that it can be directly applied to the vaccine sample without the need for desorption, dissolution or chemical reactions. The nondestructive and non-invasive nature of IF are maybe the most important features of the assay, which are essential for the implementation of Process Analytical Technology (PAT) tools and to support Quality by Design (QbD) approaches [42,43]. The PAT concept actually aims at understanding the processes by monitoring quality attributes in real time [43]. As such, there is a need for non-destructive methods of quantification that can be applied in/ on-line to increase testing efficiency and consistency, while also reducing over-processing and number of rejects. It is highly conceivable that the IF method can be adapted for in-process monitoring of antigen concentration during vaccine manufacturing using in-line fluorimeters [44]. In conclusion, we have described a direct, non-destructive, simple and inexpensive fluorimetric assay for the determination of protein concentration in vaccines containing commonly used aluminum salt adjuvants. The method exhibited high accuracy, sensitivity and reproducibility within the protein concentration range found in most vaccines. The application of this technique is not limited to the determination of protein concentration in aluminum adjuvant containing vaccines, as it has great potential for the quantification of proteins formulated with other types of adjuvant systems, such as cationic peptide-CpG, squalene emulsions, saponins, liposomes, etc. Studies looking at the applicability of the IF method to these adjuvant systems are underway. Acknowledgements The authors would like to express their cordial thanks to Michelle Nieuwesteeg for technical assistance in the preparation of this manuscript. Disclosure of potential conflict of interest During the period in which this work was performed all authors of this manuscript were Sanofi Pasteur’s employees.
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Please cite this article in press as: Nouchikian L et al. An intrinsic fluorescence method for the determination of protein concentration in vaccines containing aluminum salt adjuvants. Vaccine (2018), https://doi.org/10.1016/j.vaccine.2018.08.005