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The study demonstrates a systematic approach to stabilize PBS-formulated mAbs against freeze-thaw degradation.
Article submitted: Oct. 20, 2015
Article accepted: Apr. 6, 2016
While phosphate-buffered saline (PBS) is commonly used in research and early development, significant freeze-thaw instability of PBS-formulated biotherapeutics has been established; crystallization of dibasic sodium phosphate is known as a major driver for degradation. The authors have identified an approach to stabilize PBS-based formulation for a monoclonal antibody (mAb). The effect of sodium chloride, nonionic surfactant, protein concentration, polyols, and freezing rates on freeze-thaw stability of a mAb was studied in 12 PBS-based formulations using six different freezing protocols. In formulations not containing polyols, aggregation, the primary degradation pathway, was observed for all freezing protocols, except freezing at -20 °C. Statistical analysis indicated high salt concentration as the most significant destabilizing factor followed by slow freezing at 1 °C/min, while higher protein concentration and polysorbate-80 had a stabilizing effect. Formulations containing polyols displayed no increase in aggregation for all freezing protocols. The antibody formulated in PBS containing polyols demonstrated stability on freeze-thaw stress at higher concentrations of both sodium chloride and protein. However, for polyol-containing formulations with low concentrations of both protein and salt, an increase in % polydispersity (due to submicron particle formation) was found by dynamic light scattering. The study demonstrates a systematic approach to stabilize PBS-formulated mAbs against freeze-thaw degradation.
Achieving biotherapeutic stability after freeze-thaw is an important deliverable of formulation development studies (1, 2). Routine practice in bioprocessing involves long-term storage of drug substance and of reference standard under frozen conditions to avoid chemical degradation (1, 3, 4). In addition, many protein reagents used in diagnostics and various assays in biotechnology are stored frozen and are often subjected to multiple freeze-thaw cycles during their lifetime (5).
A number of studies have been conducted to understand the parameters that affect freeze-thaw stability of protein solutions. Phosphate-buffered saline (PBS) is a common physiological buffer used in the discovery phase of formulating protein solutions in in-vitro and in-vivo preclinical studies. The use of PBS, however, is avoided for biopharmaceuticals by formulation scientists because of protein freeze-thaw instability (6-8). The observed degradation of biotherapeutics after freezing and thawing in PBS is primarily attributed to a high propensity of sodium dibasic phosphate to crystallize, exacerbated by a pH drop of up to two pH units (8). Stabilization of proteins against aggregation after freezing and thawing by surfactant has been reported and believed to be because of the alleviation of interfacial protein denaturation at liquid-ice and liquid-air inter-faces (9-11). While surfactants significantly mitigated aggregation on freeze thaw (9-11), a more complex effect, dependent on surfactant concentration, has also been observed (10). The effect of freezing and thawing rates on protein stability has been documented as well, where fast freezing/fast thawing has often resulted in minimal aggregation (6, 12). One study, however, observed a highly detrimental effect of flash freezing on the bioactivity of a model protein (13), while a recent study observed the most beneficial combination of fast freezing and slow thawing for preserving the activity of an enzyme at low concentrations (14). The effects of freezing and thawing rates are primarily attributed to the increase in solute concentration and the subsequent freeze concentrate effect on a protein’s folded structure.
Given the common use of PBS in preclinical studies and the occasional selection of PBS in early clinical studies and most frozen diagnostic reagents, the authors conducted a systematic analysis of the freeze-thaw stability profile of a monoclonal antibody (mAb) formulated in PBS as a function of the vehicle composition and freezing protocol to identify potential stabilizing agents that would enable degradation-free frozen storage in PBS-based formulations. During the course of a three-part study, the authors assessed the stability profile of the mAb formulations after a single freeze-thaw stress (Part I), studied the long-term frozen storage stability of the formulation (Part II), and generated a mechanistic perspective of the observed effects using differential scanning calorimetry (Part III).
The mAb used in the investigation is a recombinant mAb manufactured in Chinese hamster ovary (CHO) cells at Morphotek, Inc. and formulated in PBS (10 mM Na phosphate, 150 mM NaCl, pH 7.2). The formulation matrix and freezing protocols are listed in Table IA and B, respectively. Sample containers were 2.0-mL round bottom sterile cryogenic polypropylene vials. Samples for slow freezing and thawing protocols (P2 and P4) were placed in a CoolCell cell-freezing container (Biocision) for a highly reproducible (1 °C/min) cooling rate. Samples subjected to flash freezing (P3 and P5) were loaded into plastic cryogenic boxes and immersed into liquid nitrogen for 3-5 min prior to placement into a -80 °C freezer.
A single freeze-and-thaw process was conducted by freezing the samples overnight (12 h) and thawing for 8 h. All samples were thawed at 2-8 °C, except for samples that were flash frozen in liquid nitrogen vapor, which were thawed at ambient temperature (18-25 °C) to combine fast freezing with fast thawing. Product quality was analyzed by size-exclusion high-performance liquid chromatography (SE-HPLC) for soluble aggregates and dynamic light scattering (DLS) for submicron particulates. Duplicate samples were used for each protocol and each formulation. Analyses were initiated upon thawing and completed within two weeks, during which samples were stored at 2-8 °C.
The data were analyzed for significant effects using the screening model of the statistical software package JMP 10 (2012 SAS Institute) to identify the orthogonal factors that have a large statistically significant impact on the response (15). The level of aggregation was the response. The sum of three high molecular weight (HMW) species HMW1, HMW2, and HMW3 was used for the total soluble aggregate content. Relative aggregation, which was used as the response in the screening model, refers to the ratio of percentage aggregates post-freeze/thaw to percentage aggregates before freezing.
Protein concentration, salt concentration, presence and type of polyols, freezing protocols, and presence of surfactant were the factors that were screened for effect on the response. Tonicifier content was denoted as 0, 1, and 2 for polyol-free, sucrose, and sorbitol in the formulations, respectively.
The screening results report “contrast values” (16), where the larger the contrast value of a parameter, the more that parameter affects aggregation. A bar chart plots the t-ratio, which is “contrast value” divided by pseudo-standard error. The larger the t-ratio, the greater the impact. Simultaneous p-value is analogous to the standard p-value for a linear model, but is adjusted to multiple comparisons (16). Significant parameter estimates were identified when p-value was <0.001.
The SE-HPLC chromatogram of the studied mAb formulated in PBS displays three HMW species (see Figure 1). For this study, data analyses were conducted on the sum of three HMW species (HMW1, HMW2, and HMW3). Molecular weights of different association states of the mAb were evaluated by multi-angle light scattering (MALS) detection, which showed dimers for HMW2, hexamers for HMW1, and higher-order aggregates (approximately 24-25 monomeric IgG1 units) for HMW3. The HMW3 contribution to the total HMW content was drastically lower than that of HMW2 (see Figure 1).
Freeze-thaw stress Figure 2 shows the level of soluble aggregates assessed by SE-HPLC before and after freeze-thaw stress test. An increase in aggregates was observed after a single freeze-thaw in all formulations, except those containing polyols (formulations F9, F10, F11, and F12) and F8 with low salt and low protein content. The data demonstrate significantly higher aggregation with a slower freezing than on flash freezing. For comparative analysis, the ratio of the post- to pre-freeze-thaw aggregate levels was modeled in the JMP 10 software to identify factors that significantly affect stability. The results of screening analysis are given in Table II, showing the significant effects of freezing protocol, NaCl concentration, protein concentration, and P-80 concentration, with the highest contrast values, established as orthogonal factors and p-values at or below 0.01. Interactions between freezing protocol and NaCl concentration, freezing protocol and tonicifier, as well as freezing protocol and protein concentration were also observed. The screening results were consistent with the observed experimental trends, where the most detrimental factor was the slow freezing protocol (P2) and the highest level of NaCl, while polyols eliminated aggregation on freeze thaw. The screening modeling shows statistical significance of the experimentally observed effects.
An explanation for the detrimental effect of P2 (slow freezing and thawing rate 1°C/min) might be a longer residence time at higher protein and excipient concentrations in the freeze-concentrate than that which might occur by more rapid freezing at -80 °C in P1 or flash freezing in P3. The beneficial effect of a lower NaCl concentration could be related to a lower salt concentration in the freeze-concentrate, while the beneficial effect of the higher protein concentration may be attributed to the smaller percentage of denaturation/degradation at the boundaries of the freeze-concentrate and other potential complex multiphase interfaces induced by freezing. A lesser protective effect of polysorbate-80 could be indicative of physical degradation occurring predominantly in the freeze-concentrate rather than at the ice-liquid and/or ice-air interfaces. The protective effect of polyols is in line with preferential exclusion of the polyols from the protein surface and enhanced hydration, as described by Arakawa and Timasheff (17). Select studies showed that sucrose in the range of 1%-5% and sorbitol in the range of 1%-3% prevented the aggregation of mAb after a single freeze thaw, so fine-tuning of polyol concentration can be easily incorporated into development of PBS-based formulations.
Surprisingly, no aggregation was observed on freezing at -20 °C (P6), while long-term storage at -20 °C was the most detrimental frozen storage condition (18). Since the aggregates of this antibody are not reversible at 2-8 °C, the two-week window during which analyses were conducted cannot be the reason for not seeing aggregation of the single -20 °C freezing/thawing process. Additionally, the SEC analysis was conducted consistently within the first few days after thawing, hence the erratic aggregation observed only over long-term storage of the antibody at -20 °C cannot be explained by the analysis window. It is possible that the solution super-cooled at -20 °C and did not freeze at baseline, hence generating similar results to P7 (liquid storage at 2-8 °C). It is more likely that super-cooling was accompanied by slow crystallization of NaCl, thus preventing aggregation on freeze-thaw stress by a kinetic effect. This hypothesis is in line with the long-term storage at -20 °C, where erratic aggregation was observed (18). Storage at -20 °C is near NaCl crystallization temperature as measured by DSC (19), thus protein aggregation is likely due to denaturation at the crystallized salt interface. The results for protocol P6 demonstrate limitations of freeze-thaw stress testing alone as a predictor of protein stability during long-term frozen storage at temperatures close to that of excipient crystallization or glass transition.
Submicron particles in the tested formulations were estimated by DLS. The DLS parameters calculated in secular mode with data fitted to a single mode distribution (monomer) generated % polydispersity (% Pd), a measure of the heterogeneity of the scattering particulates. A higher % Pd corresponds to a higher heterogeneity of the particle pool and hence a higher percentage of larger particles. The % Pd values for the freeze-thaw stressed samples are summarized in Figure 3. There was no effect of freezing on % Pd for any formulation or freezing protocol. Interestingly, formulations 11 and 12 containing low protein and NaCl concentrations in the presence of polyols displayed the largest % Pd even prior to freezing, whereas formulations F9 and F10 containing a polyol in the presence of high protein and high NaCl concentrations had comparable % Pd as other formulations. The reason for such difference is not known; formulation F6 under protocol P2 also showed as high % Pd as F12, which was thought to be due to the presence of foreign submicron particles in that sample preparation.
The data suggest that addition of polyols to low protein and low NaCl containing formulations favors submicron particle formation, even when aggregation as detected by SE-HPLC is completely blocked. The results of the regularization fit (where the DLS data are fit to multiple pools of different types of particles) show that this higher heterogeneity in the low salt and low protein polyol-containing F11 and F12 samples is likely associated with a small number of large particles since most of the DLS signal intensity is correlated with monomeric antibody as reflected by negligibly low % intensity and % mass of the larger particles. As expected, there was no change in protein concentration of these formulations by absorbance at 280 nm. These results point to the necessity to complement analysis of aggregation with evaluation of submicron and subvisible particles using orthogonal methods such as DLS, microscopic flow imaging, and light obscuration (20). The mAb used in this study exhibited very low levels of subvisible particles throughout formulation development, hence analysis by methods such as microscopic flow imaging was not conducted because of material limitations. The data presented in the current study are consistent with the results published earlier on the specifics of freeze-thaw aggregation and particulate formation in phosphate-buffered formulation (21).
Overall, this study presents an approach for systematic evaluation of the effect of vehicle composition and freezing protocol on the stability of antibodies upon freezing and thawing. A sequel study (Part II) describes long-term frozen storage of the same PBS-based formulations when frozen under the same freezing protocols as described in this study (18). Analysis of both data sets together with the glass transition temperatures of the tested formulations (19) provide an understanding of the kinetic and thermodynamic processes involved in protein degradation upon frozen storage and identification of conditions for optimal long-term frozen storage stability.
The use of PBS as a physiologically suitable protein formulation was enabled by addition of polyols to overcome aggregation upon a single freeze-thaw condition. The most detrimental conditions leading to the IgG instability after freeze thaw were high NaCl concentration and slow freezing and thawing rates. On the contrary, increasing the protein concentration as well as addition of polysorbate-80 tended to improve freeze-thaw stability of PBS-based formulations. This systematic approach to optimizing PBS-based formulations could have applications in a broad range of biotherapeutics and life-science reagents.
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Vol. 29, No. 8 Pages: 33-39
When referring to this article, please cite as T. Mezhebovsky et al., "Enabling Freeze-Thaw Stability of PBS-Based Formulations of a Monoclonal Antibody,"
40 (8) 2016.