NTA: Protein Aggregation

The NTA as a rapid and information-rich multi-parameter nanoparticle characterization technique which allows the user to obtain number frequency particle size distributions of polydisperse nanoparticulate systems has resulted in its rapid adoption as an interesting new technique in a wide range of sectors within the pharmaceutical sciences. This Chapter addresses some of the latest work in the literature in which NTA has been proposed, used and assessed in the study of protein aggregation and in the characterization of virus preparations and viral vaccine products.

Sub-micron particles in proteinaceous products

The subject of therapeutic protein aggregates has been studied in depth for many years and Arakawa has comprehensively reviewed the area in a series of papers covering general aspects of the mechanisms of aggregate formation and analysis (Arakawa et al., 2006), the use of analytical centrifugation and dynamic light scattering (Arakawa et al., 2007a) and FFF (Arakawa et al., 2007b) in aggregation analysis while Krishnamurthy discussed emerging technologies for analysis of protein production (Krishnamurthy et al., 2008).

The detection of microcontamination, specifically non-soluble particulates such as aggregates in liquid formulations (historically known as parenteral solutions but which are now described as injectable solutions or injectables) are proscribed by national legislation (e.g. as laid out by US (USP), European (EP), and Japanese (JP) Pharmacopoeia standards). While the limits were based on the original counting efficiencies of available technology (e.g. USP22 test <788> as 10,000 counts per container at 10 μm and 1,000 counts per container at 25 μm) the recent revision of USP 23 <788> re-defines these limits as 6,000 counts per container at 10 μm and 600 counts per container at 25 μm (United States Pharmacopoeia, 2011).

The importance of detection and enumeration of sub-visible particles (down to 100nm in diameter) in therapeutic protein products has recently been the subject of much debate. Carpenter has suggested that the lack of understanding and the clinical significance of overlooking such particles may compromise product quality. He concluded that subvisible protein particles have the potential to negatively impact clinical performance to a similar or greater degree than other degradation products, such as soluble aggregates and chemically modified species that are evaluated and quantified as part of product characterization and quality assurance programs and that current USP particulate testing is not designed to control the potential risk of large protein aggregates to impact protein immunogenicity. Analytical methods that can assess particulate characteristics (including composition, amount and reversibility of the protein aggregate) are critical for developing scientifically sound approaches for evaluating and mitigating risk to product quality caused by large protein aggregates. Furthermore, he advocated that pharmaceutical and academic researchers and instrument manufacturers should work together to help define the quantitative capabilities of current particle concentration measuring instruments for particles as small as 0.1µm and develop new instruments as needed (Carpenter et al.,  2009). He more recently highlighted the potential inaccurate quantitation and sizing of protein aggregates by size exclusion chromatography suggesting the use of orthogonal methods to assure the quality of therapeutic protein products was essential (Carpenter et al., 2010; Barnard et al., 2012).

During a recent workshop on protein aggregation and immunogenicity, Barnard and Carpenter (2012) reviewed analytical methods for detecting aggregates showing that NTA optimally covered a range of significant interest in this area. Carpenter’s group have most recently shown that recombinant murine growth hormone particles are more immunogenic with intravenous than subcutaneous administration, using NTA to measure, in their determination of the immune response in mice to injections of formulations of recombinant murine growth hormone (rmGH), the added controlled levels of protein particles (in addition to soluble, monomeric rmGH, the samples prepared contained either nanoparticles of rmGH or both nano- and microparticles of rmGH). No dependence of the immune response on particle size and distribution was observed but the immune response measured after the second injection was most pronounced when i.v. administration was used (Christie et al., 2014).

However, in forwarding an industry perspective on the subject, Singh has reiterated that the link between aggregation and clinical immunogenicity has not been unequivocally established; and emphasized that such particles are present in marketed products which remain safe and efficacious despite the lack of monitoring. He concluded that while measurement of subvisible particulates in the <10 µm size range has value as an aid in product development and characterization, limitations in measurement technologies, variability from container/closure, concentration, viscosity, history, and inherent batch heterogeneity, make these measurements unsuitable as specification for release and stability or for comparability at the present time. (Singh et al,. 2010).

It is clear, however, that elucidation of the potential problems associated with sub-micron contaminants and aggregates in proteinaceous products and the ability to legislate for their detection and enumeration remains hampered by lack of instrumentation of adequate sensitivity. Zölls et al. (2012) have reviewed the available analytical methods for the analysis of visible and sub-visible particles in therapeutic protein formulations and describe the underlying theory, benefits, shortcomings, and illustrative examples for quantification techniques, as well as characterization techniques for particle shape, morphology, structure, and identity. Similarly, Fuh (2011) has reported on the challenges faced by industry in developing analytical tools for protein stability and ligand interactions, measurement of protein aggregates as small as 30nm and reducing production costs in which the need to eliminate protein aggregates early during bioprocessing was emphasized.

Hamrang et al. (2013) have highlighted a current need for evolution of analytical methodologies used in profiling biopharmaceutical aggregation and suggested some of the techniques discussed require validation and application to biopharmaceuticals. While the development of such technologies has enabled high-throughput assessment of compounds, the implementation of recombinant DNA technology and large-scale manufacture of monoclonal antibodies and which have resulted in the biopharmaceutical stronghold in the therapeutic market, aggregate prediction and profiling still remains a challenge in the formulation of biopharmaceuticals due to artefacts associated with each analytical method.

Similarly, Rad-Malekshahi et al. (2013) and Wiggninhorn (2013) have also reviewed both the need and requirements of techniques capable of characterizing sub-micron particles in pharmaceutical products.

Finally, Zolls (2013) has comprehensively reviewed the subject and has identified and evaluated critical factors for protein particle analysis and applied this knowledge for the development of novel standardized protein-like particles, illustrating that it is crucial to not only comprehensively understand the techniques’ principle and limitations, but to also evaluate data from different techniques carefully in order to draw reliable conclusions

NTA as a monitor of sub-micron particulates in pharmaceutical products.

The ability of NTA to visualize, size and measure concentration of sub-micron particles has attracted the attention of numerous workers in this field and the technique has been assessed and applied to the real-time study of proteinaceous aggregates and their formation in several applications.

Thus Englesman, in his review of strategies for the assessment of protein aggregates in pharmaceutical products, concluded that NTA, as a single particle detection and characterization technique, was very useful for polydisperse samples though, compared to other techniques, it had a low sample throughput and, as an emerging technique, required trained operators (Engelsman, 2010). Similarly, Mire-Sluis et al. (2011) concluded that NTA is a useful method for the analysis of sub-micron aggregates though could be confounded by high concentration or opalescent background solutions and that while the technique could be considered promising had yet (at the time of writing) to be widely used in pharmaceutical applications while in a more recently published book on the analysis of aggregates and particles in protein pharmaceuticals (Mahler and Jiskoot 2012), a number of Chapters discuss the role that NTA can play in the quantitation and characterization of aggregates of therapeutic proteins (Carpenter et al., 2012; Zhao et al., 2012; Printz and Friess, 2012). Singh and Toler (2012) have compared a wide range of techniques, including NTA, for the monitoring of subvisible particles in therapeutic proteins.

The subject of protein particles and their detection and analysis has been concisely reviewed in two recent publications (Ripple and Dimitrova, 2012 and Das, 2012) in which it was concluded that further analytical progress is needed to better classify and characterize the diversity of particles encountered in therapeutic proteins, which may vary in the degree of protein unfolding, the inclusion of nonprotein nucleation centres and aggregate morphology.

Similarly, Barnard et al. (2012), in their characterization and quantitation of aggregates and particles in interferon-β products to investigate potential links between product quality attributes and immunogenicity, used NTA (as well as microflow imaging and resonant mass measurement) to characterize particles while aggregates were characterized and/or quantified using size-exclusion chromatography (SEC), analytical ultracentrifugation, gel electrophoresis, and dot-blotting immunoassays the results of their study strongly suggesting that protein aggregate and particle contents are key product quality attributes in a given product's propensity to elicit the production of neutralizing Abs in patients.

Precipitation of alpha chymotrypsin in the simultaneous presence of ammonium sulphate and t-butanol (three phase partitioning) resulted in preparations which showed self aggregation of the enzyme molecules (Rather et al., 2012). The presence of aggregates was confirmed by SEM and gel filtration on Sephadex G-200. While DLS reported aggregates in the range 242–1124 nm, NTA reported an aggregate range of 130–462 nm which probably reflected the sensitivity of DLS to being weighted incorrectly to the larger aggregates present.

van de Weert and Arvinte (2012) have recently discussed the use of protein aggregate-specific dyes such as Thioflavin T which is known to bind to amyloid.

Gruia (2011) described the characterization of submicron particle distributions in biological formulations and suggested that NTA was a novel technique which has the potential to enhance the current analytical capabilities for detecting, sizing and concentration measurement of particles in the sub-micron range.

In demonstrating that triethylenetetramine (TETA) prevented insulin aggregation and fragmentation during copper catalyzed oxidation, Torosantucci et al. (2013) used NTA to monitor the aggregation of insulin as part of this study concluding that TETA is a potential candidate excipient for inclusion in formulations of oxidation-sensitive proteins.

Ellison et al. (2013) developed a novel anodic particle coulometry method for agglomeration and aggregation studies based on sizing silver nanoparticles impacting a micro carbon electrode in a KCl/citrate solution. While this technique was shown to be in excellent agreement with NTA, they claimed that the electrochemical technique has the advantage of directly yielding the number of atoms per impacting nanoparticle irrespective of its shape, while they suggested NTA requires a correction for the non-spherical shape of agglomerated nanoparticles to derive reasonable information on the agglomeration state

Several other techniques have also recently been compared to NTA and were discussed in a recent book dedicated to biophysics for therapeutic protein development (Wei and Polozov, 2013; Wang et al., 2013; Struble et al., 2013).

Comparison of NTA to Dynamic Light Scattering (DLS)

The ensemble averaging technique of DLS, alternatively known as photon correlation spectroscopy (PCS), has historically been used for the detection of aggregates in proteins. As an ensemble technique it addresses very large numbers of particles but is limited in its ability to resolve polydisperse samples and suffers from being an intensity weighted technique which can be heavily biased to low numbers of larger particles. Furthermore, DLS cannot furnish information on particle concentration with any accuracy (Pecora, 1985).

Filipe et al. (2010) have critically evaluated the NTA technique for measurement of nanoparticles and protein aggregates stating that NTA was shown to accurately analyze the size distribution of monodisperse and polydisperse samples and that sample visualization and individual particle tracking were features that enabled a thorough size distribution analysis. They confirmed that the presence of small amounts of large (1,000nm) particles generally did not compromise the accuracy of NTA measurements, and a broad range of population ratios could easily be detected and accurately sized. NTA proved to be suitable to characterize drug delivery nanoparticles and protein aggregates, complementing DLS. Live monitoring of heat-induced protein aggregation provided information about aggregation kinetics and size of submicron aggregates. They concluded that NTA is a powerful characterization technique that complements DLS and is particularly valuable for analyzing polydisperse nanosized particles and protein aggregates. These findings were subsequently further discussed in more general terms by Jiskoot et al. (2011).

In their comparison of DLS and NTA for the analysis of lysozymes, Li et al. (2011) used NTA to measure the size distribution of the 100nm dense liquid clusters that exist in lysozyme solutions and that DLS overestimates the mean size of the clusters because of the sixth power dependence of the scattered light intensity on the size of the scatterers. Furthermore, the factor of overestimation depends on the shape of the size distribution and was ∼1.6x in the studied solution and the related underestimate of the cluster concentration is ∼10x. Similarly, the applicability of NTA, compared to DLS, to the monitoring of precipitation of a poorly water soluble drug was tested and found to give additional information not offered by DLS. Nanoparticle precipitation at the concentrations used was considered to be of relevance to high throughput screening in early drug discovery (Gillespie et al., 2011).

Furthermore, Gillespie (2011), in discussing his comparison of NTA and DLS when monitoring drug precipitation, showed that, in the analysis of the poorly soluble anti-fungal compound Tolnaftate, NTA was capable of generating an image of the particle’s scattering from which an estimate of particle concentration was available, while DLS could not generate concentration data nor detect changes in particle distributions or polydispersity over time.

In describing the success with which the development of poorly soluble and/or permeable drug molecules using nanocrystal formulations has proven to be highly successful, Wang et al. (2011) described not only the usual characterization techniques to determine physical properties such as DLS and SEM but also novel techniques such as NTA and dual polarization interferometry (DPI) as having recently emerged, pointing out that while NTA is based on DLS, it actually tracks the Brownian motion of nanoparticles quantitatively which enables the study of nanocrystal and stabilizer interactions

In comprehensively assessing the validity range of centrifuges for the regulation of nanomaterials: from classification to as-tested coronas, Wohlleben (2012), benchmarked analytical ultracentrifugation (AUC), DLS, hydrodynamic chromatography (HDC) and NTA against the known content of bimodal suspensions in the commercially relevant range between 20 nm and a few microns in an attempt to careful validate methods for the quantification of dispersability and size distribution in relevant media, and for the classification according to the EC nanodefinition recommendation. He stated that “the results validate fractionating techniques, especially AUC, which successfully identifies any dispersed nanoparticle content from 14 to 99.9 nb% with less than 5 nb% deviation”. He also claimed “In contrast, our screening casts severe doubt over the reliability of ensemble (scattering) techniques and highlights the potential of NTA to develop into a counting upgrade of DLS”. He further concluded that the “recently introduced technique NTA” measured intrinsically number distributions, but was not standardized, especially not for the determination of number% below a certain threshold. With NTA, they did adjust parameters for optimum conditions, knowing the expected results but would not have detected the bimodality with the same ‘blind routine approach’ that they took for DLS, HDC, and AUC. These findings were supported by a recent recommendation by the Environmental Protection Agency to use NTA for nanoparticle detection, but only when complemented by microscopic techniques. He finally pointed out that “the specific comment in the EC recommendation that the threshold is based on dividing the number of primary particles below 100 nm by the total number of primary particles. Hence, it is not sufficient to determine only the fraction below 100 nm, but the entire distribution is needed, which is a challenge for NTA”.

Matayoshi and Wang (2013) have patented published a new method employing a novel imaging and data analysis method for the detection of particles in therapeutic products comparing results to those obtained by NTA and other techniques.

Applications in antibody preparations

NTA has been used specifically for monitoring and analyzing aggregation antibody preparations. Mickisch et al. (2010) used both NTA and MicroFlow Imaging (MFI) for the analysis of sub-visible particles in a monoclonal antibody formulation (IgG at 1mg/ml) formulated in phosphate buffer (pH 7.2) exposed to agitation stress (stirring for 48 h and agitation in vials for up to 1 week) given both techniques represented new methods worthy of assessment. In contrast to light obscuration, MFI was demonstrated to have the advantage of not underestimating proteinaceous particles. NTA, in contrast to DLS, was demonstrated to be a powerful technique for the determination of unbiased particle distributions of polydisperse samples. They found that all formulations became visibly turbid after several hours of agitation. It transpired that, for NTA-analysis, all samples had to be diluted prior to the measurement and a broad distribution of aggregated species was obtained with average values between 150nm – 400nm after stirring and slightly lower values after agitation. Standard deviations were generally rather high. With DLS it was possible to follow the loss of monomer but show that the particle distributions were also broad and partly biased to larger particles as compared to NTA. Reproducibility was better than with NTA and dilution was not necessary. Nevertheless, they concluded that the two novel methods presented powerful tools for the characterization of particles providing complimentary information to existing methods (Mickisch et al., 2010).

Joubert et al. (2011) employed NTA for the classification and characterization of therapeutic antibody aggregates using multiple techniques capable of measuring percent aggregation, particle concentration measurements, size distribution, morphology, changes in secondary and tertiary structure, surface hydrophobicity, metal content, and reversibility. While they acknowledged no single technique was adequate for characterizing IgG aggregates, detection of particles in the nanometer range (20-1000 nm) for each stressed sample was achievable through NTA.

In a similarly broad study, Maddux et al. (2011) investigated multidimensional methods, including NTA, for the formulation of biopharmaceuticals and vaccines noting that determination and preservation of the higher order structural integrity and conformational stability of proteins, plasmid DNA, and macromolecular complexes such as viruses, virus-like particles, and adjuvanted antigens were often a significant barrier to the successful stabilization and formulation of biopharmaceutical drugs and vaccines. In another study of PEGylated stress induced aggregation of insulin and mono-PEGylated insulin, Torosantucci et al. (2011) employed NTA to confirm that NTA characterization showed submicron aggregates in the size range between 50 and 500 nm, concluding that PEGylation does not protect insulin against forced aggregation.

On comparing NTA with Atomic Force Microscopy (AFM) for the analysis of monoclonal antibody aggregation intermediates, Lee et al. (2010) showed that, whereas DLS is an ensemble technique that tries to recover a particle size distribution from the combined signal of all particles present in the sample, NTA investigates the diffusion of individual particles. Thus, DLS calculates the average particle diameter by measuring fluctuation in scattering intensity and is therefore highly affected by the presence of a few large particles it subsequently tends to be weighted to the larger particles sizes.. Using DLS (Coulter N4-Plus Submicron Particle Sizer) and NTA for an identical FA-TEGALA nanoprodrug, the average size calculated by DLS was 126 nm, which was larger than the size calculated by NTA (97 nm). The comparison of size distribution and average size from DLS and NTA indicate that a few larger nanoprodrugs (>300 nm) have a significant influence on the size calculation in DLS (Lee et al., 2010).

A variation of NTA has recently been described (Filipe et al., 2011) in which fluorescently labelled IgG and aggregates thereof were tracked (as well as the presence of control 100nm fluorescently labelled beads in complex formulations). They also used fluorescence NTA to analyze fluorescently labelled IgG and human serum albumin subjected to heat stress. These initial studies were expanded in later work on the immunogenicity of different stressed IgG monoclonal antibody formulations in immune tolerant transgenic mice (Filipe et al., 2012). The size, amount, morphology and type of intermolecular bonds of aggregates, as well as structural changes and epitope integrity were characterized and correlated with their immunogenic potential through analysis of anti-drug antibody (ADA) titres by bridging ELISA. Both unstressed IgG and freeze-thawed formulation did not induce measurable ADA levels.

Filipe et al. (2013a) have recently reviewed numerous analytical approaches to assess the degradation of therapeutic proteins such as murine IgG, including NTA, resonant mass measurement (RMM), electrical zone sensing (EZS) and fluorescence-activated microflow imaging. The review aimed to summarize the strengths and the pitfalls of current methods for assessing protein degradation, emphasizing the analytical challenges and discussing the most effective strategies during product development.

Additionally, Filipe et al. (2013b) have also described the in vivo fluorescence imaging of IgG1 aggregates after subcutaneous and intravenous injection in mice by fluorescently labelling a human mAb (IgG1), aggregated by agitation stress and injected in SKH1 mice through SC and IV routes. Their results showed differences in biodistribution and residence time between IgG1 aggregates and monomers. The long residence time of aggregates at the SC injection site, in conjunction with elevated cytokine levels, may have contributed to an enhanced immunogenicity risk of SC injected aggregates compared to that of monomers. NTA was used to determine the degree of aggregation of the material used.

Finally, Bell et al. (2013) have employed and compared DLS, NTA and DCS to quantify the degree of adsorption of IgG onto gold nanoparticles to better understand the behavior and fate of nanoparticles in biological systems. When the protein layer was formed completely, the results from all methods were consistent to within ~20% scatter and suggested that IgG adsorption on these 20 nm to 80 nm nanoparticles is rather similar to adsorption on flat gold surfaces with a water content of ~60% by volume. They reported that NTA and DLS provided, as expected, similar values that also correlated well with plasmon frequency shift. However, DCS analysis underestimated protein shell thicknesses in this regime and this may be explained through redistribution of the protein shell which reduces the frictional force during sedimentation.

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