Protein Particle Characterization

Aggregates not only vary by size and counts, but also by their morphology and composition including conformation and covalent modifications of protein within the particles.79,116 _The

conformation of the protein can be described by its secondary and tertiary structures and by its surface hydrophobicity, and can range from being native, partially unfolded, or extensively unfolded. Aggregates can also have various covalent linkages, some resulting from disulfide cross-linking, which are reducible, and other crosslinks that are not be reducible. Additionally, amino acid residues can be modified by chemical reactions which lead to formation of thioether and dityrosine covalent bonds, oxidation of methionines or cysteines, or deamidation reactions, etc.116 _{The morphology of protein molecules within particles and aggregates can be studied by a}

variety of techniques and can range from amorphous to fibrillar in structure. Characterizing aggregates with respect to these traits is potentially just as important as being able to size and count them. Protein particles containing many of these physicochemical aspects have been implicated in different immunogenic responses.35,127,128

Biophysical techniques such as Circular Dichroism (CD), Fourier Transform Infrared Spectroscopy (FTIR), Raman Spectroscopy, and Fluorescence Spectroscopy can be used to study conformational changes in proteins leading to aggregation. These techniques will be briefly discussed, but detailed information can be obtained from other sources.129,130

CD is a measure of the unequal absorption of right and left-handed circularly polarized light. Near-UV CD is used to monitor wavelengths between 250-350 nm and can give

information regarding the tertiary structure characteristics of a protein by monitoring certain amino acid residues. Far-UV CD can be used to study changes in secondary structure of the protein, by examining wavelengths between 170-250 nm to monitor the protein polypeptide backbone conformation, such as alpha helices, beta sheets, or random coil. However, larger protein particles or turbid solutions can give inconsistent results from absorption flattening and differential scattering effects, but recent developments are being made to look at the structure of protein within immobilized particles using a rotating cylindrical sample cell and ultrathin path length cells.119,131

Fourier Transform Infrared Spectroscopy (FTIR) analysis can provide an estimate of the amount of various secondary structure components in a protein solution by looking at the vibration characteristics of the bonds in the protein backbone, especially in the Amide I region between 1600-1700cm-1_. 79,132-134 _{The use of FTIR Microscopy allows increased sensitivity and}

makes it possible to determine the composition of protein molecules within a single protein particle 126_{. The advantage of FTIR analysis of proteins is that it can be performed in optically}

clear and turbid solutions or with solid samples. Unfortunately, the sensitivity is fairly low and a relatively large amount of aggregate needs to be present to detect changes in the protein higher order structure. Raman spectroscopy gives similar and complementary information as FTIR,

based on inelastic Raman scattering. Proteinaceous and nonproteinaceous particles can also be analyzed by Raman spectroscopy. Similar to FTIR microscopy, it also requires a lot of

aggregated sample to detect structural changes.135

Fluorescence spectroscopy relies on monitoring the emission of photons from certain high energy states to certain low energy states. It can be used to study changes in tertiary structure of proteins by monitoring changes in environment around aromatic amino acid residues, primarily Trp, but minimally near Tyr residues. This approach, which monitors the environment around aromatic residues is called intrinsic fluorescence spectroscopy. In extrinsic fluorescence spectroscopy, fluorescent dyes such as 1,8 anilinonapthalene sulfonic acid,

thioflavin T, sypro orange, nile red, or congo red, whose florescence properties change upon exposure to more apolar environments, are used to monitor changes in surface hydrophobicities or levels of aggregates in protein samples.136,137 _{The dyes, however, can also interfere with the}

aggregates present in solution and may either cause more aggregation or disrupt the aggregates present by their own binding to regions on the protein. Perhaps the best method using

fluorescence with individual protein particles employs FACS equipped with fluorescent detectors (described above). Mach et al have used this technique with a monoclonal antibody and showed that is simple to differentiate proteinaceous particles after staining them with a fluorescent hydrophobic dye.138

1.5.2.2 Covalent Modifications

SDS-PAGE is the simplest and fastest technique used to look for the presence of reducible or non-reducible covalent linkages (i.e., disulfide bonds) in protein samples. The technique uses an electric field to separate molecules based primarily on their molecular weight

by unfolding and coating protein molecules with a highly negatively charged detergent. However, the sample preparation itself (adding sodium-dodecyl sulfate solution and extensive heating) may modify the aggregates and cause non-covalent aggregates to dissociate, leading to inaccurate quantitation of the resulting bands. A similar technique can be performed using a capillary based system (CE-SDS) to determine the amount of covalent, disulfide linkages in aggregates. 139 _{It has been performed with several mAbs and used for detection of}

aggregates.140,141

Peptide mapping can also be used to determine if any chemical modifications occurred on the primary sequence of a mAb. First, the protein is treated enzymatically to produce peptide fragments which are then separated, monitored and identified using a combination of UPLC techniques in conjunction with UV and mass spectrometry. Using this method, a mAb, subjected to extended storage, was analyzed for the extent of deamidation and methionine oxidation.142 _In

another study, a mAb was subjected to mechanical, chemical, and thermal stress treatment and then analyzed for chemical modifications.78 _{It was shown that different types of aggregates}

contained varying levels of different types of chemical modifications.78

1.5.2.3 Morphology and Composition

The simplest way of obtaining morphological information is by using MFI, described above. The technique provides digital images of protein particles and calculates a variety of morphological parameters (area, intensity, equivalent circular diameter, perimeter, circularity, maximum ferret diameter, aspect ratio, and edge particles) of particles in solution. This

technique is fast and requires minimal sample preparation. For additional images, one can rely on an FTIR microscope, which often is accompanied with an optical microscope (described above).

The highest resolution images, however, and the greatest morphological information can be gained from Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM), or

Transmission Electron Microscopy (SEM).143 _{AFM can be used to obtain the surface topology of}

protein aggregates by using a small cantilever that moves directly over the surface of the sample with automatic height adjustments. The utility of the method is illustrated by a recent study with stressed mAbs.144 _{SEM can be used to visualize aggregates from a few nanometers to several}

microns in size by striking the sample with a beam of electrons, which scan the surface, giving information about the composition and topology of the sample. Sample preparation for SEM often requires coating the surface of aggregates with a conductive material, which may destroy or modify the aggregates. Transmission Electron Microscopy145 _{can also be a viable technique}

for visualizing aggregates down to nanometers in size. Unlike SEM, the electron beam directly interacts and passes through the sample to form an image. While sample preparation generally requires staining with uranyl acetate, samples can also be analyzed with minimal sample preparation, using a Cryo-electronic Microscopy (Cryo-EM).146 _{This technique also provides}

morphological information of the sample, but unlike SEM or TEM, the sample can be visualized in its native state. 147 _{Composition of protein aggregates can be obtained by performing Energy-}

dispersive X-ray spectroscopy (usually in conjunction with SEM or TEM) to obtain elemental information of protein within aggregates or particles. 126,148 _{The signal is obtained when a beam}

of electrons interacts with the sample. The beam of electrons can collide and eject electrons of different elements located in distinct energy levels to create a “hole”. When another electron, from a higher energy state, occupies the empty electron “hole,” the difference in energy to fill the position is released as an x-ray signal, measured by an energy-dispersive spectrometer. These x- ray signals emitted are characteristic of certain elements which can then be easily determined.149

It is extremely valuable to understand the uses and drawbacks for each of these techniques when interpreting data. Additionally, it is very important to always mention the method used in conjunction with the results since each technique is performed by relying on different scientific principles. Assumptions about shape, refractive index, and density of protein particles are made and using polystyrene standards for various measurement techniques,

although convenient, may not accurately reflect the comparative properties of protein particles. Currently there are no standards available that adequately mimic protein particles in terms of these parameters, even though extensive work is underway to develop them.