Article pubs.acs.org/ac
Polydispersity Characterization of Lipid Nanoparticles for siRNA Delivery Using Multiple Detection Size-Exclusion Chromatography Jingtao Zhang,* R. Matthew Haas, and Anthony M. Leone Department of Pharmaceutical Sciences, Merck Research Laboratories, Merck & Co., Inc., West Point, Pennsylvania 19486, United States S Supporting Information *
ABSTRACT: The development of lipid nanoparticle (LNP) based small interfering RNA (siRNA) therapeutics presents unique pharmaceutical and regulatory challenges. In contrast to small molecule drugs that are highly pure and well-defined, LNP drug products can exhibit heterogeneity in size, composition, surface property, or morphology. The potential for batch heterogeneity introduces a complexity that must be confronted in order to successfully develop and ensure quality in LNP pharmaceuticals. Currently, there is a lack of scientific knowledge in the heterogeneity of LNPs as well as highresolution techniques that permit this evaluation. This article reports a size-exclusion chromatography (SEC) method that permits the high-resolution analysis of LNP size distribution in its native solution condition. When coupled with multiple detection systems including UV−vis, multi-angle light scattering, and refractive index, on-line characterization of the distributions in size, molecular weight, and siRNA cargo loading of LNPs could be achieved. Six LNPs with sizes in the rang of 60−140 nm were evaluated and it was found that the SEC separation is efficient, highly reproducible, and can be broadly applied to a diverse range of LNPs. A comparison between the current SEC method and asymmetric field flow fractionation (FFF) shows that the current method provides similar size distribution results on LNPs compared to FFF. Two representative LNPs with similar bulk properties were evaluated in-depth using the SEC method along with two other sizing techniquesdynamic light scattering and cryo-TEM. Profound differences in batch polydispersity were observed between them. Despite the similarity in the particle assembly process, it was found that one LNP (A) possessed a narrow size and molecular weight distribution while the other (B) was polydisperse. The present results suggest that LNP drug products are highly complex and diverse in nature, and care should be taken in examining and understanding them to ensure quality and consistency. The method developed here can not only serve as a method for understanding LNP product property, permitting control on product quality, but also could serve as a potential manufacturing method for product purification. Understandings obtained in this work can help to facilitate the development of LNPs as a well-defined pharmaceutical product.
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endosomal escape, which is generally achieved by using positively charged lipids and lipid assemblies.7,8 The size properties of nanoparticles are critical to its biodistribution and performance. Generally, large sized nanoparticles (>200 nm) will be cleared rapidly to organs such as lung, liver, or spleen while small sized particles ( 10 nm) has strong angular-dependence and this behavior has long been used to calculate average radius of gyration (Rg) and molecular weight of polymers during an off-line analysis. In recent years, coupling of an in-line MALS detector to a separation technique such as SEC was shown to dramatically improve understanding in MW and size distribution of polymers, protein therapeutics, and nanoparticles.24,25 Importantly, MALS could be used to obtain absolute molecular weights and therefore eliminate the need for calibration standards in SEC.24 We examined the size and molecular weight of LNP A SEC fractions using an 18-angle light scattering detector coupled with a RI detector for concentration determination (Figure 3c−f). It is shown that rms radius (Rg) and molar mass of nanoparticles in SEC fractions decrease with the increase in elution time (Figure 3c,e). This strongly suggests that the separation of LNP A on G6000PWxl-CP is
broader size range, G6000PWxl-CP was used in all later experiments. Characterization of the Size, Molar Mass, and siRNA loading Distribution in LNP A Using a SEC-UV-MALS-RI System. To further characterize the properties of LNPs, several in-line detectors including a UV−vis diode array detector (UV), a MALS detector, and a differential refractive index (RI) detector were connected sequentially to the SEC column in the order SEC-UV-MALS-RI. It was shown before that the use of multiple in-line detectors could improve the understanding of particle heterogeneity.12,13,16 Figure 3a showed an overlay of the UV 260, LS 90°, and RI chromatogram of LNP A separated on the G6000PWxl-CP column. UV 260 was selected for being the maximum of siRNA absorbance and used as a convenient method for monitoring LNPs containing siRNA. Because of the strong contribution of particle scattering to UV signal, UV 260 alone should not be used directly to quantify siRNA concentration in LNPs.23 Instead, RI detector, a universal detector, was used to measure the weight concentration of LNPs. Light scattering detector was used to measure the size and molecular weight of the nanoparticles and its signals are proportional to both the particle concentration and their size (or molecular weight). It is shown that both the RI and the LS chromatogram closely resemble the profiles monitored at UV 260, suggesting that UV 260 could be used to roughly estimate the polydispersity of LNP A in the absence of the other detectors. It is noted that the three chromatograms appear to slightly offset each other in elution time; for example, LS signal reaches its peak the earliest while RI signal reaches its peak the latest. These data show that the earlier fractions of LNPs have relatively higher scattering responses compared with the particles that elute later, which suggest that earlier particles are larger in size. These results are consistent with the SEC separation mechanism. The use of an in-line UV−vis diode array detector permits a real-time acquisition of UV−vis spectra for each SEC fraction. UV−vis spectra or the wavelength-dependent light transmission of a nanoparticle is highly dependent on particle sizes, while the presence of a characteristic RNA absorbance peak at 260 nm could be used to verify the presence of siRNA in a nanoparticle.23 To understand the potential differences in the amount of RNA inside nanoparticles, we compared the UV 6093
dx.doi.org/10.1021/ac3007768 | Anal. Chem. 2012, 84, 6088−6096
Analytical Chemistry
Article
Figure 5. Separation and characterization of LNP B on the G6000PWxl-CP column eluted at 0.5 mL/min. (a) An overlay of the UV 260, LS 90°, and RI chromatogram of LNP B; (b) UV spectra of SEC fractions at 16, 17, 18.5, and 20 min elution time point; (c) rms radius of LNP B as a function of elution time; (d) cumulative and differential distribution of rms radius of LNP B; (e) molar mass of LNP B as a function of elution time; (f) cumulative and differential distribution of molar mass of LNP B.
based on a size-exclusion mechanism. Both the radius and molar mass of the LNP particles cover a broad range. For example, the rms radius of LNP A ranges from 19 to 55 nm, while the molar mass ranges from 3 × 107 to 4 × 108 g/mol. Using the RI detector to determine the weight concentration of nanoparticles, size distribution and molar mass distribution of nanoparticles could be calculated. The distribution analysis shows that the majority of LNP A is relatively uniform in size and molar mass. For example, 80% of LNP A particles are in a narrow rms radius and molar mass range (19−25 nm, (3−6) × 107 g/mol). On the basis of the distribution profile, the calculated weight-averaged rms radius of the particles is 23.2 nm and the weight-averaged molar mass of the particle is 5.1 × 107 g/mol. It is noted that root-mean-square radius represents a size weighted by the mass distribution about its center of mass and its value depends on the shape of the nanoparticles. For solid spheres, its value will be significantly smaller than the geometric radius or the hydrodynamic radius determined from DLS. Characterization of LNP A Size Distribution by Asymmetric FFF. To further verify the accuracy and
resolution of the separation in SEC, we employed an orthogonal separation technique, asymmetric FFF, to test the particle size distribution of LNP A. FFF was previously used for the separation of lipid and polymer based DNA delivery nanoparticles and proved to be of high resolution.12,13 Here, an asymmetric FFF system was coupled with in-line MALS and RI detectors (FFF-MALS-RI) to obtain the LNP size distribution. Figure 4a shows the light scattering and RI signal after the asymmetric field flow fractionation. Similar to the separation in SEC-MALS-RI (Figure 3), a single elution peak as well as a strong offset in the RI and LS signal is observed for LNP A, suggesting the presence of size polydispersity. From the in-line MALS signal, rms radius of the nanoparticles could be calculated (Figure 4a). It is shown that particle size increases as a function of elution time (Figure 4a). This is because, during FFF separation, smaller particles elute first due to their faster diffusion to the center of the channel where flow velocity is higher.12 On the basis of the rms radius and particle concentration data, cumulative and differential size distribution of LNP A could be obtained (Figure 4b). A comparison of the cumulative and differential size distribution between SEC 6094
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different size distributions, LNPs A and B have similar bulk chemical compositions and are processed by the same process. They only differ in their amino lipid component. The results here suggest that LNPs could be highly variable, and care should be taken in examining and understanding LNP drug products to ensure their quality and consistency. Comparison of the size distribution data from DLS, cryoTEM, and SEC-MALS-RI shows that the average sizes obtained from DLS correlate well with the data from SEC separation. However, the distribution data from DLS on these nanoparticles are of low resolution. For example, although LNP B is of much higher polydispersity than LNP A, similar polydispersity index and size distribution profiles are observed in DLS (Supporting Information Figure S2b). In contrast, the polydispersity level observed in cryo-TEM appears to be more consistent with the observations in SEC-MALS-RI. Therefore, although cryo-TEM data might not be capable of providing accurate size and size distribution data, its observations could serve as a trigger for more quantitative size separation methods described here.
(Figure 3) and FFF (Figure 4) separation shows that the distributions are almost identical to each other. On the basis of the distribution profile, the calculated weight-averaged rms radius of the particles by FFF is 23.7 nm and matches well with the data from SEC (23.2 nm). Taken together, these data strongly suggest that the separation resolution by SEC is comparable to FFF separation and reflect the size distribution in the LNPs. It is noted that FFF could offer larger dynamic range and is more versatile in its usage. On the other hand, SEC offers significant advantage in shorter method development time, is more compatible with the current pharmaceutical testing environment, and could be scaled-up for further purification or fractionation study. SEC Characterization of the Size, Molar Mass, and siRNA Loading Distribution in LNP B. We further evaluated the SEC separation of LNP B using the G6000PWxl-CP column and the in-line detectors (Figure 5). Similar to LNP A and other LNPs in Figure 2, a broad elution peak is observed for LNP B as well as a strong offset in the RI, LS, and UV 260 signal (Figure 5a), suggesting the presence of size polydispersity in LNP B. Interestingly, the offset between RI and LS is much greater in the later elution time (after 18 min) compared with LNP A, suggesting the presence of large amounts of smallsized particles in LNP B. This result is consistent with the observation in cryo-TEM (Figure 1b,d). We analyzed the UV spectra of LNP B at four different time points on the elution peak (Figure 5b). In contrast to the relatively small differences in the spectra of different LNP A fractions, there are distinct differences observed for the spectra of LNP B fractions. For instance, spectra analysis of LNP B at 16 and 17 min show few if any features of siRNA absorbance peak (at 260 nm), suggesting the lack of siRNA in the nanoparticles of large sizes. Spectra analysis of later fractions (18.5 and 20 min) shows increasingly significant siRNA absorbance peaks (at 260 nm), suggesting that smaller particles could encapsulate higher amounts of siRNA compared with larger particles. The in-line MALS detector was used to determine the size and molecular weight distribution of LNP B (Figure 5c−f). Similar to LNP A, it is found that the rms radius and molar mass of LNP B SEC fractions decrease with the increase in elution time (Figure 5c,e), confirming that separation of LNP B is also based on a size-exclusion mechanism. LNP B is determined to have a larger weight-averaged rms radius (29.7 nm) and molar mass (9.4 × 107 g/mol) compared with LNP A, which is consistent with a larger hydrodynamic radius of LNP B by DLS. Comparison between the size and molar mass range of LNP A and B shows that LNP B covers a similar size and molar mass range as LNP A. For instance, the size of LNP B ranges from 20 to 45 nm, while LNP A ranges from 19 to 55 nm. However, from radius and molar mass distribution analysis (Figure 5d,f), it is found that LNP B was observed to be much more polydisperse than LNP A. For example, while the radius of 80% LNP A is within a narrow 6 nm span (19−25 nm), no more than 30% of any LNP B population can be found within such a span. A standard deviation (σ) analysis of the differential rms radius distribution function shows that the standard deviation of LNP B distribution (5.3 nm) is much larger than that of LNP A (3.6 nm). In addition, the molar mass polydispersity (Mw/Mn) of LNP B (1.62) is also significantly higher than that of LNP A (1.16). The exact reason for the increased size heterogeneity in LNP B is not completely understood at this point. Although it is observed that different particle assembly processes could result in particles with
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CONCLUSIONS With siRNA-LNPs advancing further for clinical applications, the quality, reproducibility, and heterogeneity of the formulations are increasingly becoming a concern from pharmaceutical and regulatory perspectives. Among all physicochemical properties of LNPs, size and size distribution are of critical importance. Although DLS and cryo-TEM could be used for particle sizing, these methods are either of low resolution or difficult to provide accurate and quantitative information. This article reports a high-resolution SEC method that permits the study of the LNP size heterogeneity in its native solution condition. We demonstrate that the method is highly reproducible and could be used to evaluate the size distributions of LNPs with sizes in the range of 60−140 nm. The coupling of SEC with in-line MALS and RI detectors could provide the size and molar mass distributions of LNPs. In addition, the use of in-line UV detectors could be used to qualitatively asses the amount of siRNA loading in the LNPs. A comparison between the current SEC method and asymmetric FFF shows that the current method provides similar size distribution results on LNPs compared to FFF. Two representative LNPs (∼80 nm) with similar bulk properties were evaluated in-depth using these methods and profound differences could be found between them. Despite the similarity in the particle assembly process, LNP A is found to have narrow size and molecular weight distribution, while LNP B is found to be polydisperse. LNP B is also observed to have more particles with less siRNA loading compared with LNP A. The findings in this paper suggest that LNPs could be highly variable, and care should be taken in examining and understanding LNP drug products to ensure their quality and consistency. The method described in this article is easy to implement and is fully compatible with the current pharmaceutical testing environment. It could serve as an efficient method to control the quality of LNP drug products. With further development, SEC methods could also serve as a purification approach for polydispersed LNPs to produce high quality products with well-defined properties. Understandings obtained in this work can help to facilitate the development of LNP as a well-defined pharmaceutical product, and contribute to the progress of RNA therapeutics. 6095
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(25) Wyatt, P. J. J. Colloid Interface Sci. 1998, 197, 9−20.
ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to acknowledge Zhong Li and Lee Klein for their helpful discussion on chromatographic separation. The authors would also like to acknowledge medicinal chemists in Merck for providing the lipids and pharmaceutical science colleagues for preparing and analyzing LNP assemblies.
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