Article pubs.acs.org/molecularpharmaceutics
Polysorbate 20 Degradation in Biopharmaceutical Formulations: Quantification of Free Fatty Acids, Characterization of Particulates, and Insights into the Degradation Mechanism Anthony Tomlinson,† Barthélemy Demeule,† Baiwei Lin,‡ and Sandeep Yadav*,† †
Late Stage Pharmaceutical Development and ‡Discovery Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, California 94080, United States ABSTRACT: Polysorbate 20 (PS20), a commonly used surfactant in biopharmaceuticals, showed degradation upon long-term (∼18−36 months) storage of two monoclonal antibody (mAb, mAb-A, and mAb-B) drug products at 2−8 °C. The PS20 degradation resulted in the accumulation of free fatty acids (FFA), which ultimately precipitated to form particles upon long-term storage. This study documents the development, qualification, and application of a method for FFA quantification in soluble and insoluble fraction of protein formulation. The method was applied to the quantification of capric acid, lauric acid, myristic acid, palmitic/oleic acid, and stearic acid in placebo as well as active protein formulations on stability. Quantification of FFA in both the soluble and insoluble fraction of mAb-A and mAb-B provided a better mechanistic understanding of PS20 degradation and the dynamics of subsequent fatty acid particle formation. Additionally, the use of this method for monitoring and quantitation of the FFA on real time storage stability appears to aid in identifying batches with higher probability for particulate formation upon extended storage at 5 °C. KEYWORDS: biopharmaceutical formulations, polysorbate 20, free fatty acids, solid phase extraction, 1-pyrenyldiazomethane (PDAM), evaporative light scattering detector (ELSD), reverse phase ultra high performance liquid chromatography (RP-UHPLC), acid dissociation constant (pKa)
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INTRODUCTION
idealized molecule, one of these polyoxyethylene chains is esterified with one fatty acid chain, which is the laurate (C12) ester. The pharmacopeial specification, however, also allows for other fatty acid esters including caprate (C12), myristate (C14), palmitate (C16), oleate (monounsaturated C18), and stearate (C18).2 It is also possible to have multiple ester substitutions on each molecule (up to four) at the ends of the polyoxyethylene chains.2,3 It has also been observed that polysorbates can contain esters of polyoxyethylene isosorbides as a reaction byproduct.3,4 Polysorbate degradation by oxidation and hydrolysis is wellknown5 and has been studied in depth in various contexts, in buffers or in the raw material. The problem of polysorbate degradation in biotherapeutic formulations, however, has surfaced recently and gathered attention in the scientific and pharmaceutical communities due to its potential impact on product quality.6−8 In a recent study, Labrenz et al. proposed the existence of a bioprocess impurity potentially hydrolyzing the ester bond in polysorbate 80 (PS80) and resulting in generation of free fatty acids (FFA).9 These degradants would
In biopharmaceutical formulations, it is common practice to include surfactants to prevent absorption of the active pharmaceutical ingredient (API) to surfaces and to form a protective barrier against undesirable interactions between the biomolecule and the air−liquid interface. These interactions with the air−liquid interface can lead to protein denaturation and aggregation.1 A commonly used surfactant for preventing these types of interactions is polysorbate 20 (PS20). This material is a complex mixture that is primarily composed of a group of molecular entities with a sorbitan headgroup bound to four polyoxyethylene polymer chains (Figure 1). In the
Received: Revised: Accepted: Published:
Figure 1. Chemical structure of polysorbate 20, w + x + y + z = 20, R = lauric acid in idealized PS20. © 2015 American Chemical Society
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April 21, 2015 September 23, 2015 September 29, 2015 September 29, 2015 DOI: 10.1021/acs.molpharmaceut.5b00311 Mol. Pharmaceutics 2015, 12, 3805−3815
Article
Molecular Pharmaceutics
mass spectrometry. Additionally, because of the large relative amounts of protein and excipients in comparison to FFAs, there may be some significant matrix effects that could make mass spectrometric quantification more challenging.17 Taking these practical considerations into account, a reverse phase ultra high performance liquid chromatography (RPUHPLC) method was developed and utilized in this study for quantification of fatty acids in protein drug product solutions and placebos. The method provides a high throughput extraction procedure coupled with a rapid and simple HPLC method for FFA quantification. Additionally, the 96-well format used for the extraction procedure makes it amenable to automation by liquid handlers. The qualified method was subsequently used to detect and quantify the FFA composition in the soluble and insoluble fractions of two liquid mAb drug products. The applicability of the method was also demonstrated in the detection and quantification of very low levels of FFA changes, which could potentially serve as a tool for early detection of PS20 degradation in formulations.
likely have limited solubility and would be prone to precipitate as visible particles in formulations. Elevated subvisible and visible particulates have been observed in two mAb (mAb-A and mAb-B) formulations after long-term (∼18−36 months) storage at 2−8 °C. A recent publication used Raman microscopy on the same two mAb formulations and showed that these particles were composed of free fatty acids.10 Both molecules (liquid drug products) also showed ∼20% PS20 degradation over 24 month storage at 2−8 °C. It then follows that the fatty acid particles observed during long-term storage are polysorbate degradation byproducts that have limited solubility at low temperatures. In addition, PS20 raw material also contains unesterified FFAs as byproducts from the manufacturing process. The presence of these FFA, from PS20 raw materials or as degradation byproducts of PS20, can potentially impact product quality if precipitated, resulting in elevated levels of subvisible and visible particulates. It is therefore critical to identify and quantify these byproducts in order to better understand the solubility limits as well as the mechanisms involved in particle formation. This information could further be crucial to developing a mitigation strategy. A major challenge limiting the development of a FFA quantification method is from matrix effects incurred by protein and excipients. Many currently available methods are labor intensive and time-consuming, commonly using a modified version of the Bligh−Dyer liquid−liquid extraction procedure.11,12 Many of these extraction methods have been shown to be reasonably accurate and precise, however, they are not easily amenable to high throughput formats and liquid handler automation. It can also be quite challenging to quantify very low (μg/mL to ng/mL) levels of fatty acids in the presence of active biopharmaceuticals. Additionally, the fatty acid molecules of interest have no chromophores or fluorophores and are thus difficult to detect spectrophotometrically. It is also difficult to chromatographically resolve the polysorbate related fatty acids and fatty acid esters due to their similar hydrophobicities. Recently, many chromatographic methods have been developed to study the ester composition of polysorbates and to quantitate intact polysorbate in protein formulations using evaporative detectors. These methods, however, might not be able to quantify fatty acids in placebo or drug product solutions due to the relatively high volatility of the fatty acids themselves.3,4,13−15 Moreover, methods using evaporative detectors would find challenges in comparing quantities of semivolatile fatty acids due to differences in response factors for fatty acids with varying volatilities. This can be seen clearly by the breadth in melting temperatures of these various fatty acids, with melting temperatures reported from 13.4 °C for oleic acid up to 69.3 °C for stearic acid.16 Another recent report described a method that could detect fatty acids in the presence of polysorbate, but this method (developed for polysorbate 80) may also have very different sensitivities to the different FFA products present in PS20 degraded samples, due to the use of an evaporative light scattering detector (ELSD).9 Although there have been efforts in our laboratory to develop a FFA quantification strategy using mass spectrometry that provides the sensitivity required for the analysis of degraded PS20 formulation samples (unpublished data), the ease of use of a UV detector provides more versatility and simplifies method transfers from site to site in the biopharmaceutical testing environment. From a pharmaceutical development perspective a method in this format would also be more amenable to method validation and the quality control environment than
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MATERIALS AND METHODS Materials. mAb-A and mAb-B (called “mAb-1” and “mAb2” respectively in a recent publication from Saggu et al. in 201510) formulated drug product vials and unconditioned bulks (no stabilizer or polysorbate) were supplied by Genentech, Inc. (South San Francisco, CA, USA). Acetonitrile and ethyl acetate (HPLC grade) were purchased from Avantor Performance Materials (Phillipsburg, NJ). Methanol (HPLC grade) was purchased from Burdick and Jackson (Morristown, NJ). Polysorbate 20 (Tween 20) was purchased from Croda Inc. (Mill Hall, PA) and was stored with a nitrogen overlay at 2−8 °C. Guanidine-HCl was obtained from EMD-Millipore (Milford, MA). 1-Pyrenyldiazomethane (PDAM) was obtained from Molecular Probes (Eugene, OR). Fatty acids were purchased in a kit from Supelco (Bellefonte, PA), individual purities: lauric acid (C12), 99.7%; capric acid (C10), 99.9%; myristic acid (C14), 99.9%; palmitic acid (C16), 99.5%; stearic acid (C18), 99.9%. The solid phase extraction (SPE) cartridges (Oasis HLB 96-well, 30 mg of sorbent per well) and the UHPLC column (Acquity BEH-300, C18, 2.1 × 150 mm) were obtained from Waters (Milford, MA). The water used in this study was purified using the Elga PureLab Ultra filtration system and had a resistivity greater than 18 MΩ cm. Polycarbonate gold filters with 0.8 μm pore size (Pall Corporation, Timonium, MD) were used for particle isolation. Sample Preparation. For assay qualification in active drug product, the unformulated/unconditioned UFDF bulk was formulated for a final mAb-A DP at 60 mg/mL protein, stabilizer, and 0.04% (w:v) PS20 at pH 5.4. The formulation buffer used during assay qualification was composed of the same excipients as the final formulated mAb-A. Protein or placebo samples containing 0.02% to 0.04% PS20 (w:v) and PS20 degradants were purified with an Oasis HLB resin in the 96-well plate format (WAT058951). The procedure roughly followed the extraction procedure described by Kim and Qiu for extraction of polysorbates.18 The resin was prewashed with 1 mL of 100% acetonitrile followed by 1 mL of purified water at −10 mmHg pressure. 500 μL of drug product or placebo sample (100−200 μg of PS20 and PS20 degradants) was then added and drawn through the resin with a pressure of −5 mmHg. The resin was then washed with 1 mL of 4 M guanidine-HCl and 2 × 1 mL of 10% methanol in purified water. The fatty acids and polysorbate were then eluted using 3806
DOI: 10.1021/acs.molpharmaceut.5b00311 Mol. Pharmaceutics 2015, 12, 3805−3815
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Figure 2. 1-Pyrenyldiazomethane and the mechanism of action for derivatization of fatty acids via the carboxylic acid headgroup.
Figure 3. Representative chromatograms showing a fatty acid standard sample at 10 μg/mL and a FFA sample extracted from formulated mAb-A, both derivatized with PDAM and analyzed by RP-UHPLC. The figure shows (a) a full view of the chromatogram and (b) a zoomed in view of the fatty acid peaks.
500 μL of 100% acetonitrile or 25% isopropanol and 75% acetonitrile (v:v), first allowing the elution solvent to be in contact with the resin for >120 s to ensure higher recovery. During development of the method, two SPE elution conditions were evaluated. Elution with 100% acetonitrile proved to be highly accurate for capric, lauric, and myristic acid, while palmitic and stearic acid showed lower recoveries. The addition of 25% isopropanol to the elution condition yielded better recoveries for stearic and palmitic acids than acetonitrile alone with no change to retention times of the FFA peaks. Samples eluted from the SPE and the standards (which were prepared directly in 25% isopropanol and 75% acetonitrile, v:v, or 100% acetonitrile) were then derivatized by adding 50 μL of 1 mg/mL PDAM in ethyl acetate to 200 μL of sample or standard. These samples were then kept in the dark, at room temperature, overnight for complete derivatization of the fatty acids in solution. This reaction, based on the work of Nimura et al., is depicted in Figure 2.19 Since the fatty acids have very low solubility in aqueous solutions, the FA standards were prepared by dissolving the FFA directly into the solvent (either acetonitrile or 25% isopropanol and 75% acetonitrile, v:v) and then derivatized
with PDAM. The FFA standards were not extracted by SPE. Additionally, preparing the standards in this manner provided a way to assess the extraction recovery by comparing extracted samples to the standards directly diluted into solvent. Chromatographic Analysis of FFA. The analysis of the derivatized fatty acids was achieved using a Waters Acquity HClass Bio ultra performance liquid chromatograph with an Acquity photodiode array (PDA) detector using an Acquity BEH-300 C18 reverse phase column (1.7 μm particle size, 2.1 mm × 150 mm) maintained at 70 °C. The compounds were separated using a gradient of >18 MΩ cm water (mobile phase A) and HPLC grade acetonitrile (mobile phase B). The gradient conditions were as follows: 0.0−2.0 min, 70% B; 2.0− 6.0 min, 70−100% B; 6.0−8.0 min, 100% B; 8.0−8.1 min, 100− 70% B; 8.1−12 min, 70% B. The flow rate was 0.5 mL min−1 with a total run time of 12 min. Detection was achieved using the PDA detector at 241 nm (the absorbance maximum wavelength of PDAM) with a bandwidth of 4.8 nm. The injection volume was 5 μL. A needle wash with 100% acetonitrile was implemented to minimize carryover. Data were analyzed using the Waters Empower 3 software package. Typical chromatograms are shown in Figure 3. 3807
DOI: 10.1021/acs.molpharmaceut.5b00311 Mol. Pharmaceutics 2015, 12, 3805−3815
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and 12.5 μg/mL. This analysis was performed in triplicate from three separate stock solutions. The assay was determined to be linear within this range for fatty acids dissolved in either 25% IPA with 75% acetonitrile (v:v) or only 100% acetonitrile, with R2 > 0.985 (Figures 5a and 5b). In order to determine the range, the residuals for the averages of the standard points were also analyzed (Figures 5c and 5d). Extraction Recovery, Accuracy, and Precision. Extraction recovery and accuracy were assessed by spike recovery in both drug product and placebo. A 500 μg/mL solution of lauric acid was prepared in acetonitrile and diluted 1:100 in either formulation buffer or in the mAb-A formulation with 60 mg/ mL protein. These two samples were each tested in triplicate and compared to the drug product and the placebo solutions before the spike. The samples were also tested for FFA concentration following SPE by a previously qualified mass spectrometry method for quantification of fatty acids in PS20 raw material (unpublished data, manuscript in preparation) to confirm the results from the PDAM UHPLC method (Table 1). Because the PS20 raw material itself contains some fatty acids (particularly lauric acid), the expected concentrations of the spiked samples were calculated by the following formula:
Mass Spectrometric (MS) Quantification of FFA. Mass spectrometry analysis was performed using a Waters Acquity UPLC with a Waters LCT Premier XE mass spectrometer. The fatty acids were first separated on a Waters BEH C18 column prior to MS analysis utilizing direct electrospray ionization (ESI) in negative ion mode with time-of-flight (TOF) detection. The assay was qualified for use at Genentech for PS20 raw material characterization. (Unpublished data, manuscript in preparation.) Polysorbate 20 Quantification. Polysorbate 20 quantification was performed by mixed-mode chromatography with evaporative light scattering detection as described previously by Hewitt et al.13 PS20 quantification was performed by this method in all subsequent sections.
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RESULTS AND DISCUSSION Specificity, Linearity, and Assay Range. The specificity of the assay was assessed by dissolving 5 μg/mL of capric (C10), lauric (C12), myristic (C14), palmitic (C16), oleic (monounsaturated C18, C18-1), and stearic (C18) acids in acetonitrile individually, derivatizing each sample with PDAM, and separating them by RP-UHPLC. Figure 4 shows that
final spiked sample conc ⎡ = ⎢(sample vol) × (sample conc) ⎢⎣ ⎛ (spike vol) × (spike conc) ⎞⎤ × ⎜1 + ⎟⎥ /final vol (sample vol) × (sample conc) ⎠⎦ ⎝
From the expected concentration of the spiked sample, a spike recovery was calculated using the following formula: % spike recovery = 100 ×
exptl spiked sample conc expected spiked sample conc
For other fatty acids, a similar strategy was employed, except that a single solution containing 200 μg/mL of each of the five fatty acids being assayed (capric, lauric, myristic, palmitic, and stearic acids) was prepared in 25% isopropanol and 75% acetonitrile (v:v) and then subsequently diluted 1:100 in formulation buffer or 60 mg/mL of formulated mAb. The spiked buffer and formulated mAb samples were then compared to the unspiked controls, and the recoveries for each fatty acid were calculated. In all spike recovery samples, the recoveries were between 89.7−125.2% (Table 2). In some extreme cases of polysorbate degradation, it may be necessary to dilute the extracted fatty acid samples from the SPE to prepare a sample that has FFA concentrations within the established assay range, however, this has not been observed in testing thus far. Interassay precision was assessed by assaying two samples six times each over two assay preparations. The samples were prepared by spiking mAb-A formulation buffer and mAb-A DP spiked to final concentrations of 2.5 μg/mL capric, lauric, and myristic acids, 1.25 μg/mL palmitic acid, and 0.6 μg/mL stearic acid. The assay was shown to have a maximum relative standard deviation (RSD) of 9.9% for both samples tested (Table 3). The assay precision for capric, lauric, and myristic acids was relatively tight with maximum RSD 4.7%, whereas palmitic/ oleic and stearic acids showed a maximum RSD of 9.9%. The spiked samples contained lower concentrations of palmitic and stearic acids, due to their reduced solubility in placebo or DP.
Figure 4. Injections of individual fatty acids derivatized with PDAM and analyzed.
capric, lauric, myristic, and stearic acids could be baseline separated from all the other fatty acids tested, however palmitic and oleic acids overlap in their elution profile. It also appears that there are some fatty acid contaminants, particularly palmitic or oleic acids and stearic acid, that are present in the blank and that could be related to impurities in the solvents or C18 column being used (Figure 4). However, the baseline signal for these contaminants remained constant for all samples and with multiple injections. These offsets to the signals were represented and corrected for within the calibration curves for C16/C18-1 and C18. The assay was assessed to be linear in the range of 0.5 μg/mL to 12.5 μg/mL for all five of the fatty acids tested. In order to determine the linearity and range of the assay, a stock solution containing capric (C10), lauric (C12), myristic (C14), palmitic (C16), and stearic (C18) acids in methanol, each dissolved at 10 mg/mL concentration, was prepared. This stock solution was prepared by weighing 250 mg of each fatty acid into a single 25 mL volumetric flask and adding methanol to 25 mL final volume. The stock solution was then serially diluted to prepare standards in the range of 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 3808
DOI: 10.1021/acs.molpharmaceut.5b00311 Mol. Pharmaceutics 2015, 12, 3805−3815
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Figure 5. (a) Linearity of five saturated fatty acids in 25% isopropanol and 75% acetonitrile (v:v), n = 3 preparations, and (b) linearity of five saturated fatty acids in acetonitrile, n = 3 preparations; (c, d) residuals for the averages of the standard points.
PDAM derivatized fatty acid peaks were then integrated manually. The standard deviations for the integrated amounts in each FFA peak region were calculated from the integrated peak areas from the six injections. The LOD was defined by multiplying the standard deviation of the noise for each peak region by a factor of 3. Any peak with an area greater than three times the standard deviation of the noise would give >99% confidence that the peak detected was related to the presence of the FFA being assayed for. Using this, the LOD for each FFA was identified, and approximate FFA concentrations corresponding to those peak areas (back calculated from a standard curve prepared in the established assay range) are listed in Table 4. Based on the assessment for specificity, linearity, accuracy, precision (interassay precision, single analyst), and limit of detection, the assay was identified to be suitable for quantification of FFA in placebo and active mAb formulations. FFA Extraction in Samples with Visible Particles. In an attempt to determine the total amount of FFA in a DP vial that had developed particulates, additional studies were conducted to test the efficiency of the resin to recover the fatty acids present as particulates and as the soluble fraction in the solution. In these experiments, a mAb-A drug product that showed elevated subvisible and visible particles after long-term
Table 1. Spike Recovery for Lauric Acid (n = 3) Measured after Extraction by the PDAM-UHPLC Method and Mass Spectrometrya analysis method
sample
SPEPDAMUPLC
formulation buffer A
MS
formulation buffer A
formulated mAb-A
formulated mAb-A a
sample type
av (μg/ mL)
SD
% CV
neat spike neat spike neat spike neat spike
1.02 5.64 0.94 6.29 0.93 5.75 0.82 6.17
0.09 0.31 0.01 0.19 0.01 0.11 0.02 0.07
8.9 5.6 1.3 3.0 1.5 1.9 2.5 1.1
% recovery 93.9 106.0 97.1 108.2
Rounded to one decimal place.
Therefore, the high variability for those FFAs was likely caused by the low concentrations of these fatty acids, which were measured at the lower end of the assay range. The assay limit of detection (LOD) was also assessed by injecting six injections of a blank of 0.2 mg/mL PDAM in a solution containing 20% ethyl acetate, 20% isopropanol, and 60% acetonitrile. The peak regions that corresponded to the 3809
DOI: 10.1021/acs.molpharmaceut.5b00311 Mol. Pharmaceutics 2015, 12, 3805−3815
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Molecular Pharmaceutics Table 2. Spike Recovery of FFA Spiked into DP and Placeboa sample
fatty acid
formulation buffer A neat
capric acid lauric acid myristic acid palmitic/oleic acid stearic acid capric acid lauric acid myristic acid palmitic/oleic acid stearic acid capric acid lauric acid myristic acid palmitic/oleic acid stearic acid capric acid lauric acid myristic acid palmitic/oleic acid stearic acid
formulation buffer A spiked (2 μg/mL of each FFA)
formulated mAb-A neat
formulated mAb-A spiked (2 μg/mL of each FFA)
N = 1 (μg/ mL)
N = 2 (μg/ mL)
N = 3 (μg/ mL)
av (μg/ mL)
SD
NDb 1.41 0.53 0.52
ND 1.42 0.53 0.49
ND 1.48 0.55 0.56
N/Ac 1.44 0.54 0.52
N/A 0.04 0.01 0.04
N/A 2.96 2.57 6.74
N/A N/A N/A N/A
0.25 2.48 3.33 2.44 2.26
0.25 2.51 3.37 2.47 2.42
0.28 2.51 3.37 2.46 2.41
0.26 2.50 3.36 2.46 2.37
0.02 0.02 0.03 0.02 0.09
7.27 0.67 0.78 0.65 3.78
N/A 125.2 97.9 97.0 94.1
1.79 ND 0.98 0.43 0.48
2.10 ND 0.95 0.45 0.52
2.19 ND 0.99 0.44 0.53
2.03 N/A 0.97 0.44 0.51
0.21 N/A 0.02 0.01 0.03
10.40 N/A 2.16 1.62 5.51
89.7 N/A N/A N/A N/A
0.24 2.60 3.30 2.59 2.67
0.25 2.31 2.94 2.31 2.43
0.32 2.29 2.93 2.28 2.40
0.27 2.40 3.05 2.39 2.50
0.04 0.17 0.21 0.17 0.15
15.58 7.21 6.88 7.30 5.97
N/A 120.0 103.1 98.2 99.8
2.40
2.27
2.21
2.29
0.10
4.35
101.1
% RSD % spike recovery
a
The values were rounded to two decimal places. N = 1, N = 2 and N = 3 are three separate replicates for this experiment. In order to calculate spike recovery for some fatty acids, values outside of the assay range (0.5−12.5 μg/mL) were utilized. bND: not detected. cN/A: not applicable.
Table 3. Interassay Repeatability for mAb-A DP and mAb-A Formulation Buffera sample
FFA
N=1
N=2
N=3
N=4
N=5
N=6
av
SD
RSD (%)
spiked formulation Buffer A
capric lauric myristic palmitic/oleic stearic capric lauric myristic palmitic/oleic stearic
2.77 3.62 2.76 1.29 0.48 2.87 2.84 2.56 1.37 0.51
2.88 3.82 2.92 1.40 0.56 2.71 2.70 2.45 1.31 0.47
2.94 3.83 2.94 1.52 0.61 2.76 2.70 2.32 1.21 0.48
2.93 3.92 3.01 1.42 0.63 2.89 2.84 2.39 1.18 0.50
3.06 4.04 3.08 1.39 0.56 2.84 2.80 2.34 1.14 0.48
3.04 4.04 3.09 1.48 0.63 3.10 2.99 2.49 1.20 0.49
2.94 3.88 2.97 1.42 0.58 2.86 2.81 2.42 1.23 0.49
0.11 0.16 0.12 0.08 0.06 0.14 0.11 0.09 0.09 0.02
3.7 4.1 4.1 5.7 9.9 4.7 3.9 3.8 7.0 3.1
FFA spiked in mAb-A
a
Replicates N = 1, N = 2, and N = 3 were from the same assay, and replicates N = 4, N = 5, and N = 6 were from a second, separate assay.
A sample, the vial was gently swirled to resuspend any settled particles. During the extraction of this sample, the flow-through of each wash step was collected. The flow-through and washes were then reanalyzed using SPE extraction and derivatization to determine whether the presence of fatty acid particulates affects the retention and extraction of fatty acids in the sample. From the data in Figure 6, it was observed that particles minimally affected the recovery of fatty acids by the SPE. The lack of fatty acids in the flow-through and washes indicates that the initial extraction was sufficient to extract both the soluble fraction of FFA and the fatty acids contained within particles. FFA Content in Soluble and Insoluble Fractions of mAb Drug Products Showing PS20 Degradation. The qualified FFA quantification method was thereafter utilized to analyze the FFA content in DP bulk as well as in visible particles in a mAb-A formulation after >3 years of storage and a mAb-B formulation after >5.5 years of storage at 2−8 °C. The visible particles in 8 mL of each drug product were filtered through a 0.8 μm pore size, gold-coated polycarbonate filter. To
Table 4. LOD for Each FFA Was Determined and Approximate Concentrations from LOD Peak Area Values Were Calculated from a Standard Curve Prepared within the Established Assay Rangea FFA capric lauric myristic palmitic/ oleic stearic a
SD of peak area (μV s)
3 × SD of peak area (μV s)
approx LOD conc (μg/mL)
682.9 37.3 180.7 492.7
2048.6 111.9 542.0 1478.1
0.3 0.3 0.3 0.2
55.4
166.3
0.1
Approximate LOD concentrations are rounded to one decimal place.
storage at 2−8 °C (∼36 months) was extracted by SPE, derivatized with PDAM, and analyzed by RP-UHPLC. These vials were routinely checked during manufacturing for particles, and none were observed. Before pipetting out 500 μL of mAb3810
DOI: 10.1021/acs.molpharmaceut.5b00311 Mol. Pharmaceutics 2015, 12, 3805−3815
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Figure 6. Amounts of FFA in the SPE elution versus the amounts of FFA lost in the flow-through (FT) and the various wash steps in formulated DP containing particles.
prevent any redissolution of the insoluble fraction during filtration, the vials were kept cold on ice during sampling. After filtration, the retentate was rinsed with ice-cold water to remove any proteinaceous or FFA remnants from soluble fraction on the filter. The insoluble fraction on the filter was then resuspended in 1 mL of methanol. The dissolved particles were then derivatized with PDAM and analyzed to determine the FFA composition of the particles. Additionally, the same DP samples were passed through the SPE, derivatized with PDAM, and analyzed to determine the total FFA (soluble and insoluble) in each drug product vial. These same samples, following extraction or filtration, were additionally analyzed by liquid chromatography mass spectrometry (LC−MS) as further verification of the method. The relative amounts of the various FFA in the drug product vials, as well as the composition of the filtered particles, are tabulated in Table 5 and shown in Figures 7 and 8. A very good agreement was observed for FFA content quantified using the PDAM derivatized method and the LC− MS analysis, further validating the suitability of this method for FFA quantification. This analysis also showed that although lauric acid represents the largest proportion of the total FFA content in solution as well as in neat PS20 (as an ester), the insoluble particles are enriched with longer chain (>C12) fatty acids. Lauric acid, in particular, is in significantly lower proportion in the particles when compared to bulk (Figures 7 and 8). This indicates that the particle formation is largely driven by the solubilities of different fatty acids (longer chain fatty acids having lower solubility in aqueous solutions) along with their relative abundance in the original PS20 mixture. Even though the longer chain fatty acids represent a lower fraction of the heterogeneous PS20, their hydrophobicities suggest that, in a sample with ∼20−30% PS20 degradation, there would be amounts of longer chain FFA likely to exceed solubility limits, nucleate, and form particles in biopharmaceutically relevant aqueous solutions. This would also suggest that it would be prudent to consider the percentage of the fatty acid ester chains in raw PS20 materials and, if available, preferentially select PS20
Table 5. Percentage of Each FFA in Solution and in Particles from Different Lots of DP Compared to the Composition of Fatty Acids in Filtered Particles from DP Vialsa FFA conc (μg/ mL)
sample
fatty acid
mAb-A SPE (total in solution and in particles)
mAb-B SPE (total in solution and in particles)
sample filtered mAb-A particles
filtered mAb-B particles
SPEPDAMRPUHPLC
LC− MS
% of total FFA by SPE-PDAM-RPUHPLC method (%)
0.2 9.6 4.9 1.9
2 55 28 12
0.3 0.3 6.1 3 1.3
3 3 49 24 15
0.5
4
capric 0.3 lauric 8.9 myristic 4.4 palmitic/ 2.0 oleic stearic 0.5 capric 0.3 lauric 6.0 myristic 2.9 palmitic/ 1.8 oleic stearic 0.4 FFA conc (μg/8 mL)
fatty acid capric lauric myristic palmitic/ oleic stearic capric lauric myristic palmitic/ oleic stearic
SPEPDAM-RPUHPLC
LC− MS
% of total FFA by SPEPDAM-RP-UHPLC method (%)
0.3 2.8 4.4 3.2
0 2.9 4.7 3.1
2 25 39 29
0.5 0.3 1.4 6.7 4.8
0.3 0 1.2 6.4 3.7
5 2 10 48 35
1.1
1
8
a
The mAb-A sample was >3 years old, and the mAb-B sample was >5.5 years old.
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DOI: 10.1021/acs.molpharmaceut.5b00311 Mol. Pharmaceutics 2015, 12, 3805−3815
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Figure 7. Chromatogram of mAb-A DP containing particles characterized by extracting fatty acids, derivatizing with PDAM, and analysis by RPUHPLC and particles from the same vial filtered on a 0.8 μm gold filter, resuspended in methanol, derivatized with PDAM, and analyzed by RPUHPLC.
Figure 8. Comparison of relative amounts of various fatty acids in the total solution versus the amounts in filtered particles in (a.) mAb-A and (b.) mAb-B.
reported previously that the pKa of fatty acids increases with increase in fatty acid chain length. The pKa of the carboxylic acid group is ∼4.5; however, for longer fatty acids like palmitic and stearic acids, pKa values >8 have been reported.20 The pKa value for lauric acid has been reported from 5.321 in water to 6.6 in the presence of nonionic C12 polyoxyethylene glycol surfactant n-dodecyloctaoxyethyleneglycomonoether at concentrations above its CMC.22 Assuming a pKa of lauric acid around ∼6.0, the ionized fraction and hence the solubility of lauric acid in mAb-B formulation would be higher due to higher formulation buffer pH. This is reflected in the composition of the insoluble particles for mAb-A and mAb-B, wherein mAb-B particles show lower lauric acid content owing to higher solubility in this formulation. On the other hand, for myristic, palmitic, oleic, and stearic acids, the pKa will be much higher. The pKa’s of myristic acid and palmitic acid have been reported
lots containing lowest levels of long chain fatty acid esters such as palmitates, oleates, and stearates. Comparing the insoluble particle composition in mAb-A and mAb-B DP is also informative. The particles formed in mAb-B DP show lower lauric acid content and somewhat higher myristic and palmitic acid contents when compared to the particles formed in mAb-A DP. These differences are likely related to the pH and PS20 content in the formulation. The total solubility of the fatty acid will be a sum of the ionized and un-ionized fraction. The ionized fraction will be governed by the pKa of the fatty acid and the pH of the solution, whereas the solubility of the un-ionized fraction will be influenced by the PS20 concentration that will aid in solubilization of the unionized fatty acid in the hydrophobic micellar core. The mAb-A formulation is at pH 5.4 with 0.04% (w:v) PS20 whereas mAbB is formulated at pH 6.0 with 0.02% (w:v) PS20. It has been 3812
DOI: 10.1021/acs.molpharmaceut.5b00311 Mol. Pharmaceutics 2015, 12, 3805−3815
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Molecular Pharmaceutics
Figure 9. FFA tested in mAb-A DP stability samples stored at 5 and 25 °C. These samples were tested for FFA in samples frozen at the designated time points for (a) capric, (b) lauric, (c) myristic, (d) palmitic/oleic, and (e) stearic acids.
as 8.1−8.2 and 8.6−8.8, respectively, in water.23 To the best of our knowledge the pKa of myristic, palmitic, and higher chain fatty acids in the presence of a C12 based nonionic surfactant is unavailable in the literature. However, based on the previous observations that the pKa of fatty acids will increase with chain length, and assuming that pKa’s of these long chain fatty acid are ≥7.0, these FFA moieties will predominantly be protonated and therefore show very little difference in solubility between mAb-A and mAb-B buffers. In such instances, the amount of PS20 (approximately two times more in mAb-A than mAb-B) in the buffer will have a solubilizing effect. The effect of the pKa has been studied in depth and is described further in a forthcoming manuscript from Doshi et al.24 This is further reflected from the particle composition analysis in mAb-A and mAb-B formulations in which myristic, palmitic/oleic, and stearic acids constitute a lower fraction in mAb-A particles due to their increased solubility with the higher PS20 concentration in the formulation. The amount of lauric acid quantified in both the mAb-A and mAb-B DP samples using this method correlates very well with the amount of PS20 degraded. The formation of lauric acid by a hydrolytic mechanism would give 1:1 stoichiometry between the degradation of monolaurate PS20 and the formation of lauric acid. The pharmacopeial specification for laurate ester content in PS20 is 40−60%.2 Assuming a laurate ester content of 50%, 0.04% (w:v) PS20 as in mAb-A would have approximately 0.163 mM polysorbate with lauric acid esters. Assuming that these lauric acid esters are monolaurate PS20, ∼25% PS20 degradation in mAb-A sample will generate approximately 0.05 mM (8.2 μg/mL) lauric acid, which is consistent with the experimentally determined value of 8.9 μg/ mL lauric acid in mAb-A sample. The mAb-B stability sample showed ∼31% degradation of PS20 after >5.5 years. Similar theoretical calculations would predict a lauric acid concentration of 5.4 μg/mL in the mAb-B sample, which is very close to the experimentally determined value of 6.0 μg/mL.
To further elucidate the capability and applicability of this method, the change in PS20 and FFA content was monitored in mAb-A formulation placed on stability at 5 and 25 °C. The changes in capric, lauric, myristic, palmitic/oleic, and stearic acid content over 18 months are plotted in Figure 9. For most of the FFA, and particularly lauric acid (which is the most common fatty ester of PS20), a significant increase of FFA could be observed as early as 1 month when stored at 5 °C. This trend of increasing FFA concentration was observed for all FFA under both storage conditions. Additionally, by tracking the degradation products, the PS20 degradation can be detected sooner compared to change in PS20 content. When compared with data from an established PS20 quantitation assay,13 at the 1 month time point when stored at 5 °C, an increase of ∼30−40% is observed in fatty acid concentrations, whereas the concentration of PS20 takes 6 months to drop by more than 10% (Figure 10). This shows that the assay described herein can provide an early indication for identifying batches with a high propensity for PS20 degradation even at low (5 °C) storage temperatures. Having this information would give a strong indication of which lots are most likely to develop FFA related particulates over long-term storage. Although these instances of PS20 degradation have been observed in some liquid mAb formulations as discussed here, there is currently no root cause identified. In all likelihood, there may be a few mechanisms that could be acting to various degrees in different products. Oxidation and hydrolysis are the two mechanisms primarily responsible for polysorbate degradation.25 In a hydrolytic mechanism, two major classes of degradation products can be formed, the FFA and the sorbitan POE headgroup. On the other hand, oxidative degradation results in generation of fatty acid esters and various other degradants such as aldehydes, acids, and ketones. The oxidative degradation may also result in generation of trace amount of fatty acids (data not shown). The FFA generated from hydrolysis or oxidation will tend to have lower solubility in 3813
DOI: 10.1021/acs.molpharmaceut.5b00311 Mol. Pharmaceutics 2015, 12, 3805−3815
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Molecular Pharmaceutics
products of PS20. Both mAb-A and mAb-B formulations showed an increase in FFA content upon real-time storage, and concomitantly showed appearance of visible particulates. Analysis of visible particulates indicated that there is a preferential enrichment of longer chain FFAs in precipitated particles. It is likely that due to their lower solubilities the longer chain FFAs (such as myristic, palmitic, and stearic acids) drive nucleation and precipitation of fatty acid particles in DP compared to shorter chain FFAs. Additionally the differences in mAb-A and mAb-B particulate composition provide evidence that the FFA solubilities are also influenced by the formulation compositions, e.g., pH and polysorbate content. FFAs are the major products of the hydrolytic degradation of polysorbates.25 In both mAb-A and mAb-B formulations, the amount of PS20 degraded and the amount of lauric acid formed was stoichiometrically equivalent. This, along with evidence from previous studies into polysorbate degradation,25 gives a strong indication that the major mode of degradation in the mAb-A and mAb-B formulations is hydrolytic in nature. It is highly unlikely that the FFA levels generated in mAb-A and mAb-B formulations are due to chemical hydrolysis given the extremely slow hydrolysis rates at these formulation pHs and storage at 2−8 °C.25,27 Although a causative agent has not been identified, these data suggest that the degradation observed in these cases may be catalyzed by an impurity in the DP, a possibility proposed by Labrenz in 2014.9 In conclusion, the method and approach presented in this work to monitor FFA levels in products where polysorbate degradation is observed can provide an early detection of the issue as well as knowledge into the mechanism of degradation which could be subsequently leveraged for devising timely mitigation strategies.
Figure 10. A comparison of mAb-A DP stability samples tested for PS20 concentration and the same stability samples tested for lauric acid concentration.
aqueous buffers based on their partition coefficient (log P) values than their POE ester counterparts. Some of the POE ester oxidation products, however, could also have low solubility resulting in nucleation and particle formation. For example, 2-hydroxyethyl myristate would have a log P value of 5.29, and myristic acid itself would have a log P value of 5.98.25 This would mean that both of these would be largely insoluble (log P > 4) in water, but that myristic acid would be more insoluble. The method described in this study is able to accurately identify and quantify fatty acids in protein formulations. However, one caveat of this method is that it is unable to detect the POE esters of fatty acids that would be produced by an oxidative PS20 degradation mechanism. An argument can be made that in cases where a PS20 loss is observed without an increase in FFA concentration, the likely mechanism for PS20 degradation is oxidative. This needs to be further substantiated, however, since the stability of the fatty acid esters and their propensity to autohydrolyze is unknown. Nonetheless, the method described in this study provides a tool for detecting FFA that may be present in the formulations, either from raw material or as PS20 degradants in placebo and active DP, enabling early detection of polysorbate degradation products that may contribute to particulate formation. The use of such a method would not necessarily replace a method for quantification of PS20; rather, it could serve as a complementary tool under specific polysorbate instability situations.
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AUTHOR INFORMATION
Corresponding Author
*LSPD Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. Phone: (650) 225 8912. Fax: (650) 742-1504. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to thank Nidhi Doshi on her insights into fatty acid solubilities as a function of pH and Kai Zheng for help with FTIR identification of FFA particles. The authors would also like to acknowledge Purnendu Nayak for his work on the development of an LC−MS based method for FFA determination in raw materials. Additionally, we would like to thank Karen Vigeant for providing us with the mAb-B sample containing particles and John Wang for his insightful helpful support and discussions.
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CONCLUSION This work has led to new insights into mechanism of PS20 degradation over long-term storage in two of the mAb formulations via monitoring of soluble and insoluble FFA content as PS20 degradants. A high throughput method was developed to quantify FFA in placebo and active protein formulations. Similar diazomethane dye based methods have been previously used for quantification of FFA in the polysorbate raw material.7,26 To our knowledge, however, this is the first method utilizing these dyes that has been applied to absolute quantification of free fatty acids in formulated mAb samples and formulation buffer placebos containing PS20. This method was applied to track FFA as hydrolytic degradation
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ABBREVIATIONS USED API, active pharmaceutical ingredient; PS20, polysorbate 20; PS80, polysorbate 80; FFA, free fatty acids; mAb, monoclonal antibody; ELSD, evaporative light scattering detector; RPUHPLC, reverse phase ultra high performance liquid chromatography; DP, drug product; PDAM, 1-pyrenyldiazomethane; SPE, solid phase extraction; MS, mass spectrometry; RSD, relative standard deviation; LOD, limit of detection; LC− MS, liquid chromatography mass spectrometry; log P, partition coefficient 3814
DOI: 10.1021/acs.molpharmaceut.5b00311 Mol. Pharmaceutics 2015, 12, 3805−3815
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(20) Kanicky, J. R.; Shah, D. O. Effect of Degree, Type, and Position of Unsaturation on the pKa of Long-Chain Fatty Acids. J. Colloid Interface Sci. 2002, 256, 201−207. (21) Nyrén, V.; Back, E. The Ionization Constant, Solubility Product and Solubility of Abietic and Dehydroabietic Acid. Acta Chem. Scand. 1958, 12, 1305−1311. (22) Söderman, O.; Jönsson, B.; Olofsson, G. Titration of Fatty Acids Solubilized in Cationic and Anionic Micelles. Calorimetry and Thermodynamic Modeling. J. Phys. Chem. B 2006, 110, 3288−3293. (23) Kanicky, J. R.; Poniatowski, A. F.; Mehta, N. R.; Shah, D. O. Cooperativity Among Molecules at Interfaces in Relation to Various Technological Processes: Effect of Chain Length on the P Kaof Fatty Acid Salt Solutions †. Langmuir 2000, 16, 172−177. (24) Doshi, N.; Demeule, B.; Yadav, S. Understanding Particle Formation: Solubility of Free Fatty Acids as Polysorbate 20 Degradation Byproducts in Therapeutic Monoclonal Antibody Formulations. Mol. Pharmaceutics 2015, DOI: 10.1021/acs.molpharmaceut.5b00310. (25) Kishore, R. S. K.; Kiese, S.; Fischer, S.; Pappenberger, A.; Grauschopf, U.; Mahler, H. C. The Degradation of Polysorbates 20 and 80 and Its Potential Impact on the Stability of Biotherapeutics. Pharm. Res. 2011, 28, 1194−1210. (26) Siska, C. C.; Pierini, C. J.; Lau, H. R.; Latypov, R. F.; Fesinmeyer, R. M.; Litowski, J. R. Free Fatty Acid Particles in Protein Formulations, Part 2: Contribution of Polysorbate Raw Material. J. Pharm. Sci. 2015, 104, 447−456. (27) Bates, T. R.; Nightingale, C. H.; Dixon, E. Kinetics of Hydrolysis of Polyoxyethylene (20) Sorbitan Fatty Acid Ester Surfactants. J. Pharm. Pharmacol. 1973, 25, 470−477.
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