Attribute Analytics Performance Metrics from the MAM Consortium Interlaboratory Study.

The multi-attribute method (MAM) was conceived as a single assay to potentially replace multiple single-attribute assays that have long been used in process development and quality control (QC) for protein therapeutics. MAM is rooted in traditional peptide mapping methods; it leverages mass spectrometry (MS) detection for confident identification and quantitation of many types of protein attributes that may be targeted for monitoring. While MAM has been widely explored across the industry, it has yet to gain a strong foothold within QC laboratories as a replacement method for established orthogonal platforms. Members of the MAM consortium recently undertook an interlaboratory study to evaluate the industry-wide status of MAM. Here we present the results of this study as they pertain to the targeted attribute analytics component of MAM, including investigation into the sources of variability between laboratories and comparison of MAM data to orthogonal methods. These results are made available with an eye toward aiding the community in further optimizing the method to enable its more frequent use in the QC environment.

Industry White Paper: Contemporary Opportunities and Challenges in Characterizing Crystallinity in Amorphous Solid Dispersions.

Members of the IQ Consortium ″Working Group on Characterization on Amorphous Solid Dispersions″ shares here a perspective on the analytical challenges, and limitations of detecting low levels of crystalline drug substance in amorphous solid dispersions (ASDs) and associated drug products. These companies aim to employ highly sensitive commercially available analytical technologies to guide development, support control strategies, and enable registration of quality products. We hope to promote consistency in development and registration approaches and guide the industry in development of "characterization best practices" in the interest of providing high quality products for patients. The first half of this perspective highlights the unique challenges of analytical methodologies to monitor crystalline drug substance in ASDs and their associated drug products. Challenges around use of limit tests, analyte spiking experiments, and method robustness are also underscored. The latter half describes the merits and limitations of the diverse analytical "toolbox" (such as XRPD, NIR and DSC), which can be readily applied during development and, in some cases, considered for potential application and validation in the commercial QC setting when necessary.

New Peak Detection Performance Metrics from the MAM Consortium Interlaboratory Study.

The Multi-Attribute Method (MAM) Consortium was initially formed as a venue to harmonize best practices, share experiences, and generate innovative methodologies to facilitate widespread integration of the MAM platform, which is an emerging ultra-high-performance liquid chromatography-mass spectrometry application. Successful implementation of MAM as a purity-indicating assay requires new peak detection (NPD) of potential process- and/or product-related impurities. The NPD interlaboratory study described herein was carried out by the MAM Consortium to report on the industry-wide performance of NPD using predigested samples of the NISTmAb Reference Material 8671. Results from 28 participating laboratories show that the NPD parameters being utilized across the industry are representative of high-resolution MS performance capabilities. Certain elements of NPD, including common sources of variability in the number of new peaks detected, that are critical to the performance of the purity function of MAM were identified in this study and are reported here as a means to further refine the methodology and accelerate adoption into manufacturer-specific protein therapeutic product life cycles.

Subunit mass analysis for monitoring multiple attributes of monoclonal antibodies.

RATIONALE: Multi-Attribute Methods (MAMs) are appealing due to their ability to provide data on multiple molecular attributes from a single assay. If fully realized, such tests could reduce the number of assays required to support a product control strategy while providing equivalent or greater product understanding relative to the conventional approach. In doing so, MAMs have the potential to decrease development and manufacturing costs by reducing the number of tests in a release panel. METHODS: In this work, we report a MAM which is based on subunit mass analysis. RESULTS: The MAM assay is shown to be suitable for use as a combined method for identity testing, glycan profiling, and protein ratio determination for co-formulated monoclonal antibody (mAb) drugs. This is achieved by taking advantage of the high mass accuracy and relative quantification capabilities of intact mass analysis using quadrupole time-of-flight mass spectrometry (Q-TOF MS). Protein identification is achieved by comparing the measured masses of light chain (LC) and heavy chain (HC) mAbs against their theoretical values. Specificity is based on instrument mass accuracy. Glycan profiling and relative protein ratios are determined by the relative peak intensities of the protein HC glycoforms and LC glycoforms, respectively. Results for these relative quantifications agree well with those obtained by the conventional hydrophilic interaction liquid chromatography (HILIC) and reversed-phase LC methods. CONCLUSIONS: The suitability of this MAM for use in a quality control setting is demonstrated through assessment specificity for mAb identity, and accuracy, precision, linearity and robustness for glycan profiling and ratio determination. Results from this study indicate that a MAM with subunit mass analysis has the potential to replace three conventional methods widely used for mAb release testing including identification assay, glycosylation profiling, and ratio determination for co-formulated mAbs.

Development of a liver-targeted siRNA delivery platform with a broad therapeutic window utilizing biodegradable polypeptide-based polymer conjugates.

The greatest challenge standing in the way of effective in vivo siRNA delivery is creating a delivery vehicle that mediates a high degree of efficacy with a broad therapeutic window. Key structure-activity relationships of a poly(amide) polymer conjugate siRNA delivery platform were explored to discover the optimized polymer parameters that yield the highest activity of mRNA knockdown in the liver. At the same time, the poly(amide) backbone of the polymers allowed for the metabolism and clearance of the polymer from the body very quickly, which was established using radiolabeled polymers to demonstrate the time course of biodistribution and excretion from the body. The fast degradation and clearance of the polymers provided for very low toxicity at efficacious doses, and the therapeutic window of this poly(amide)-based siRNA delivery platform was shown to be much broader than a comparable polymer platform. The results of this work illustrate that the poly(amide) platform has a promising future in the development of a siRNA-based drug approved for human use.

An in vivo evaluation of amphiphilic, biodegradable peptide copolymers as siRNA delivery agents.

A series of amphiphilic, biodegradable polypeptide copolymers were prepared for the delivery of siRNA (short interfering ribonucleic acid). The molecular weight (or polymer chain length) of the linear polymer was controlled by reaction stoichiometry for the 11.5, 17.2, and 24.6 kDa polypeptides, and the highest molecular weight polypeptide was prepared using a sequential addition method to obtain a polypeptide having a molecular weight of 38.6 kDa. These polymers were used to prepare polymer conjugate systems designed to target and deliver an apolipoprotein B (ApoB) siRNA to hepatocyte cells and to help delineate the effect of polymer molecular weight or polymer chain length on siRNA delivery in vivo. A clear trend in increasing potency was found with increasing molecular weight of the polymers examined (at a constant polymer:siRNA (w/w) ratio), with minimal toxicity found. Furthermore, the biodegradability of these polymer conjugates was examined and demonstrates the potential of these systems as siRNA delivery vectors.

Assessing the heterogeneity level in lipid nanoparticles for siRNA delivery: size-based separation, compositional heterogeneity, and impact on bioperformance.

A primary consideration when developing lipid nanoparticle (LNP) based small interfering RNA (siRNA) therapeutics is formulation polydispersity or heterogeneity. The level of heterogeneity of physicochemical properties within a pharmaceutical batch could greatly affect the bioperformance, quality, and ability of a manufacturer to consistently control and reproduce the formulations. This article studied the heterogeneity in the size, composition, and in vitro performance of siRNA containing LNPs, by conducting preparative scale fractionation using a sephacryl S-1000 based size-exclusion chromatography (SEC) method. Eight LNPs with size in the range of 60-190 nm were first evaluated by the SEC method for size polydispersity characterization, and it was found that LNPs in the range of 60-150 nm could be well-resolved. Two LNPs (LNP A and LNP B) with similar bulk properties were fractionated, and fractions were studied in-depth for potential presence of polydispersity in size, composition, and in vitro silencing, as well as cytotoxicity. LNP A was deemed to be monodisperse following results of a semipreparative SEC fractionation that showed similar size, chemical composition, in vitro silencing activity, and cytotoxicity across the fractions. Therefore, LNP A represents a relatively homogeneous formulation and offers less of a challenge in its pharmaceutical development. In contrast, LNP B fractions were shown to be significantly more polydisperse in size distribution. Interestingly, LNP B SEC fractions also exhibited profound compositional variations (e.g., 5 fold difference in N/P ratio and 3 fold difference in lipid composition) along with up to 40 fold differences in the in vitro silencing activity. The impact of LNP size and formulation composition on in vitro performance is also discussed. The present results demonstrate the complexity and potential for presence of heterogeneity in LNP-based siRNA drug products. This underscores the need for tools that yield a detailed characterization of LNP formulations. This capability in tandem with the pursuit of improved formulation and process design can lead to more facile development of LNP-based siRNA pharmaceuticals of higher quality.

Polydispersity characterization of lipid nanoparticles for siRNA delivery using multiple detection size-exclusion chromatography.

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 high-resolution 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 techniques-dynamic 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.

Challenges in the pharmaceutical development of lipid-based short interfering ribonucleic acid therapeutics.

INTRODUCTION: Harnessing RNA interference as a therapeutic approach has the potential to significantly expand the druggable target space, offering new hope for treatment of diseases that cannot be addressed with existing classes of drugs. A number of siRNA therapeutics have already progressed into preclinical and clinical development. Of these, lipid-based systems have emerged as one of the most mature classes of systemic delivery technologies. Despite tremendous advances in development, a number of significant challenges must still be addressed to enable commercialization of a lipid-based siRNA pharmaceutical product. AREAS COVERED: This review addresses specific challenges inherent to the pharmaceutical development of lipid-based siRNA therapeutics. Focus is placed on the development of a robust manufacturing process, the setting of appropriate product specifications and controls, development of strategies to assess and ensure product stability, and the evaluation of product comparability throughout development. EXPERT OPINION: Discovering and developing a lipid-based siRNA therapeutic that can be commercialized requires engineering a particle that selectively and efficiently delivers the cargo to the target tissue and cellular compartment. The particle assembly must be strictly controlled and physical properties thoroughly characterized to successfully develop an understanding of particle attributes that influence in vivo pharmaceutical properties. Correlation of particle physio-chemical properties to product performance is the foundation for advancements in discovery and assuring quality in a commercial drug product. Although difficult, we believe these development challenges can be addressed with appropriate scientific resources and that the industry will continue to progress siRNA therapeutic candidates through clinical development.

Ion atmosphere relaxation controlled electron transfers in cobaltocenium polyether molten salts.

A room-temperature redox molten salt for the study of electron transfers in semisolid media, based on combining bis(cyclopentadienyl)cobalt with oligomeric polyether counterions, [Cp2Co](MePEG350SO3), is reported. The transport properties of the new molten salt can be varied (plasticized) by varying the polyether content. The charge transport rate during voltammetric reduction of the ionically conductive [Cp2Co](MePEG350SO3) molten salt exceeds the actual physical diffusivity of [Cp2Co]+ because of rapid [Cp2Co](+/0) electron self-exchanges. The measured [Cp2Co](+/0) electron self-exchange rate constants (k(EX)) are proportional to the diffusion coefficients (D(CION)) of the counterions in the melt. The electron-transfer activation barrier energies are also close to those of ionic diffusion but are larger than those derived from optical intervalent charge-transfer results. Additionally, the [Cp2Co](+/0) rate constant results are close to those of dissimilar redox moieties in molten salts where D(CION) values are similar. All of these characteristics are consistent with the rates of electron transfers of [Cp2Co](+/0) (and the other donor-acceptor pairs) being controlled not by the intrinsic electron-transfer rates but by the rate of relaxation of the ion atmosphere around the reacting pair. In the low driving force regime of mixed-valent concentration gradients, the ion atmosphere relaxation is competitive with electron transfer. The results support the generality of the recently proposed model of ionic atmosphere relaxation control of electron transfers in ionically conductive, semisolid materials.

Ion atmosphere relaxation and percolative electron transfer in Co bipyridine DNA molten salts.

Polypyridyl complexes of Co decorated with 350-Da polyether chains (Co(350)(2+)) form molten phases of nucleic acids when paired with DNA counterions (Co(350)DNA) or 25-mer oligonucleotides. Analysis of voltammetry and chronoamperometry of mixtures of these phases with complexes having ClO(4)(-) counterions (Co(350)(ClO(4))(2)) and no other diluent provides charge transport rates from the oxidation and reduction currents for the complexes. As the mole fraction of the Co(350)(ClO(4))(2) complex in the mixture is varied from ca. 0.25 to 1, the physical diffusion constants derived from the Co(III/II) wave increase from 1 x 10(-11) cm(2)/s to 5 x 10(-10) cm(2)/s, and apparent diffusion constants dominated by the Co(II/I) electron self-exchange increase from 1 x 10(-10) cm(2)/s to 2 x 10(-8) cm(2)/s. Pure Co(350)DNA melts, containing no Co(350)(ClO(4))(2) complex, do not exhibit recognizable voltammetric waves; DNA suppresses the Co(II/I) electron transfer reactions of Co complexes for which it is the counterion. There are therefore two microscopically distinct kinds of Co(350) complexes, those with DNA and those with ClO(4)(-) counterions, with respect to their Co(II/I) electron-transfer dynamics, leading to percolative behavior in their mixtures. The electron-transfer rates of the Co(II/I) couple are controlled by the diffusive relaxation of the ionic atmosphere around the reaction pair, and the inactivity of the bound Co complexes can be attributed to the very low mobility of the anionic phosphate groups in the DNA counterion. Substitution of sulfonated polystyrene for DNA produced similar results, suggesting that this phenomenon is general to other polymer counterions of low mobility. We conclude that the measured Co(II/I) charge transport and electron-transfer rate constants reflect more the diffusive mobility of the perchlorate counterion than the intrinsic Co(II/I) electron hopping rate.

Electron and mass transport in hybrid redox polyether melts contacted with carbon dioxide.

Films of neat metal salts with covalently attached oligoether side chains ([Co(bpy(CO(2)MePEG-350)(2))(3)](ClO(4))(2); bpy is 2,2'-bipyridine, and MePEG-350 is methyl-terminated oligomeric ethylene oxide with an average molecular weight of 350 Da) undergo marked changes in physical and electrochemical properties upon contact with CO(2). Electrochemical measurements indicate that the physical diffusion coefficient (D(PHYS)) of the Co(II) species, the observed rate constant for Co(II/I) self-exchange (k(EX)), and the physical diffusion coefficient of the perchlorate counterion (D(ClO4)) increase from 2.4 x 10(-11) to 7.0 x 10(-10) cm(2)/s, 6.8 x 10(5) to 4.5 x 10(6) M(-1) s(-1), and 3.4 x 10(-10) to 4.3 x 10(-9) cm(2)/s, respectively, as CO(2) pressure is increased from 0 to 2000 psi at 23 degrees C. A reduction in activation energy accompanies the enhancement of each of these properties over this pressure range. Increasing CO(2) pressure from ambient to 1000 psi causes the films to swell 13%, and free-volume theory explains the enhanced mass transport properties of the films. The origin of increases in electron-transfer kinetics is considered. Plots of log(k(EX)) versus log(D(PHYS)) and log(k(EX)) versus log(D(ClO4)) are both linear. This suggests that electron self-exchange is controlled by factors that also affect log(D(PHYS)) or log(D(ClO4)). One explanation is based on plasticization of the oligoether side-chain motions by CO(2) that affect ether dipole repolarization and Co complex diffusion rates. A second explanation for the changes in k(EX) is based on control of electron transfer by relaxation of counterions neighbor to the Co complexes, which is measured by D(ClO4). Both explanations represent a kind of solvent dynamics control of k(EX).