Advancements in High Throughput Biophysical Technologies

Review pubs.acs.org/molecularpharmaceutics

Advancements in High Throughput Biophysical Technologies: Applications for Characterization and Screening during Early Formulation Development of Monoclonal Antibodies Hardeep S. Samra*,† and Feng He*,‡ †

Department of Formulation Sciences, MedImmune, One MedImmune Way, Gaithersburg, Maryland 20878 Drug Product Development, Amgen Inc., 1201 Amgen Court West, Seattle, Washington 98119

‡

ABSTRACT: The formulation development of monoclonal antibodies is extremely challenging, due to the diversity and complexity contained within this class of molecules. The physical and chemical properties of a monoclonal antibody dictate the behavior of the protein drug during manufacturing, storage and clinical administration. In the past few years, the use of high throughput technologies has been widely adapted to delineate unique properties of individual immunoglobulin G’s (IgG’s) important for their development. Numerous screening techniques have been designed to reveal physical and chemical characteristics of a protein relevant to stability under production, formulation and delivery conditions. In addition, protein stability under accelerated stresses has been utilized to predict long-term storage behavior for monoclonal antibodies in the formulation. In this review, we summarize the recent advancements in the field of biophysical technology, with a specific focus on the techniques that can be directly applied to the formulation development of monoclonal antibodies. Several case studies are also presented here to provide examples of combining existing biophysical methods with high throughput screening technology in the formulation development of monoclonal antibody drugs. KEYWORDS: high throughput, biophysical technology, monoclonal antibody, formulation development

■

INTRODUCTION Monoclonal antibodies (mAbs) have quickly emerged as a popular class of protein therapeutics that target oncologic and immunologic diseases. To date, there are nearly 25 monoclonal antibody drugs approved worldwide, while more than 200 other mAbs are currently in the development pipeline.1,2 Monoclonal antibodies (mAbs) are complex protein molecules, composed of two copies of heavy and light chains with a total molecular mass of approximately 150 kDa.3 The majority of mAb polypeptide chains fold into antiparallel β-sheets adopting a Y shape, spherical three-dimensional structure in solutions4 (Figure 1). Though each of the well-defined domains, Fab, CH2 and CH3, contribute to the overall stability of the mAb,5 maintaining the global stability and improving delivery properties remain the focus of pharmaceutical development. Throughout the development life cycle, a mAb is subjected to a number of environmental stresses, such as cellular stress, low solution pH during purification, freeze−thaw stress, temperature excursions, transportation and distribution, etc. These stresses may impose physical alterations to the mAbs that may, in turn, affect the safety and efficacy of the drug product.6−9 Major physical degradation pathways include partial unfolding, aggregation, and particulation, as well as precipitation and loss to surface adsorption.3,10 The prevention or minimization of these physical degradations is one of the primary goals of mAb © 2012 American Chemical Society

formulation development. However, it is extremely time- and resource-consuming to follow the physical stability of a large number of formulations while executing long-term storage or distribution studies. As a result, high stress conditions, such as elevated temperatures, agitation, low or high solution pH, and high ionic strength, are often employed to accelerate the physical instability in order to identify conditions that provide better potential to allow a stable drug product formulation.11−13 Currently, most mAb based protein drugs are delivered via intravenous (iv) or subcutaneous (sc) administration. Iv therapy is usually provided for oncology drugs at clinical sites that possess such capability and often involves significant dilution of the product into the iv diluent. In contrast, sc injection is preferred for treatment of autoimmune and infectious diseases, and is often carried out via patient selfadministration. Higher doses are required due to limitations in half-life and binding affinity and increasing target coverage, and this has led to the need for increasing protein concentrations in Special Issue: Advances in Biophysical and Bioanalytical Protein Characterization Received: Revised: Accepted: Published: 696

August 14, 2011 January 8, 2012 January 20, 2012 January 20, 2012 dx.doi.org/10.1021/mp200404c | Mol. Pharmaceutics 2012, 9, 696−707

Molecular Pharmaceutics

Review

receptors in many cases. A successful formulation should ideally maintain the conformation and integrity of the mAb during long-term storage. While the exact three-dimensional conformation of a protein can only be revealed by high resolution techniques that obtain information on the atom level, such as X-ray crystallography and nuclear magnetic resonance spectroscopy, a number of biophysical techniques have been widely used to characterize the conformational and physical properties.17−19 Spectroscopic methods, including circular dichroism (CD), fluorescence, infrared, ultraviolet (UV) and Raman spectroscopy, are typically carried out to measure the structural properties, based on spectroscopic signals that arise from specific structural features of the protein higher order conformation.20 Differential scanning calorimetry (DSC) is a direct measurement of protein thermal stability by recording heat transitions which occur during protein unfolding by increasing temperature.21,22 Several other biophysical techniques are aimed to characterize the colloidal features of proteins. Optical density (OD) and static light scattering (SLS) at wavelengths where proteins do not exhibit specific absorbance or scattering intensity are often correlated with aggregates in solution. Dynamic light scattering (DLS) probes the dynamic behavior of a protein population, and the results directly reflect the protein diffusion pattern. Analytical ultracentrifugation (AUC) and some chromatographic techniques study the hydrodynamic properties of different protein species based on separation under centrifugal force or phase equilibrium. Biophysical techniques possess unique advantages when applied to protein therapeutics. The specificity for protein during these measurements is achieved where signals from the buffer components can be easily separated or subtracted. In addition, many biophysical measurements can be employed using the neat protein formulations, avoiding analytical discrepancies that might be caused by sample dilution. Since the launch of the first Tecan liquid handling system (Tecan Group Ltd., Switzerland) in 1985, high throughput technology has advanced tremendously in the field of life sciences. High throughput technologies are commonly referred to as equipment and methods that can prepare and/or analyze a

Figure 1. Representative high resolution structure of a human IgG1. Several domains comprise the two heavy chains (red and blue) and the two light chains (yellow and cyan). The Fab is made up of a constant (CH) and a variable (VL and VH) region from both the heavy and light chains. The two heavy chains comprise the Fc region, which contains multiple constant (CH) domains. (Image produced with PDB ID 1HZH utilizing Discovery Studio 3.0 from Accelyrs, Inc.).

order to maintain small dosing volumes necessary for patience convenience and comfort. 14,15 One way to overcome limitations of sc administration and to improve patient convenience is increasing the protein concentration in the drug product. Higher protein concentrations bring significant challenges to process and formulation development of mAbs, particularly in relation to protein solubility and viscosity.16 Identifying formulation conditions that enhance protein solubility as well as mitigate solution viscosity has been recognized as a critical component of the mAb product development.15 The efficacy of mAb drug products strongly depends on the integrity of the protein’s native structure, ensuring the proper presentation of complementary determining regions (CDRs) to facilitate antigen binding, as well as binding of the Fc region to

Table 1. List of High Throughput Biophysical Techniques Commonly Applied to Monoclonal Antibody Development technique differential scanning calorimetry

primary output and measurable

existing and potential applications in mAb development

high throughput capability

key refs

protein conformational stability

automation

22−27,29,32

differential scanning fluorimetry

protein melting/unfolding profile protein melting/unfolding

protein conformational stability

11,33,35,36,38−40

turbidity

sample turbidity

protein aggregation and particulation

intrinsic fluorescence

protein tertiary structure

protein conformation assessment

extrinsic fluorescence

protein surface hydrophobicity solution polarity sample scattering intensity

protein aggregate detection solution viscosity protein aggregate, particle and precipitate detection protein species characterization protein characterization, aggregate/particle detection protein interaction/aggregation propensity solution viscosity protein species characterization (size, charge, etc.) protein aggregation propensity, solubility

multiwell plate; automation multiwell plate; automation multiwell plate; automation multiwell plate; automation

static light scattering multiangle light scattering dynamic light scattering

high/ultra performance liquid chromatography

scattering particle size protein hydrodynamic size distribution protein diffusion diffusion of external tracer protein separation protein self- or crossinteraction

697

multiwell plate; automation automation multiwell plate; automation

automation

45,46,49,50 53,55,58 59,61−63,66 64,65 11,68,69 61,73 13,26,45,77,80 81,82,85 12,75,92 77,93−96,98 99,102−104

dx.doi.org/10.1021/mp200404c | Mol. Pharmaceutics 2012, 9, 696−707


Review

reported that mAb domains, i.e. Fab vs Fc, might display different susceptibility to different stresses, which makes DSC one of the ideal tools to be applied in formulation development.22,25,27 In addition, DSC remains the primary technique to reveal crystallization temperature, Tc, and glass transition temperature, Tg, as required to develop the freeze-dry process for lyophilized protein drugs.29,30 In order to increase throughput capability, the automated capillary DSC has been developed, which can analyze multiple samples that are placed into 96-well plates without any attendance required (MicroCal, Piscataway, NJ). This technological advancement certainly provides a new platform for screening many protein formulations over a short time course, and the method meets the high throughput demand with the capability to analyze close to 20 samples per day, depending on the scanning speed. However, a major obstacle of the current technology is its compatibility with high protein concentrations. For example, the currently available DSC settings would only accommodate mAb samples up to a concentration at about 5 mg/mL, far less than that of many approved products, as well as those in development that might exceed 100 mg/mL. It has been noticed previously that the unfolding and aggregation behavior of mAb proteins can significantly depend on protein concentration.22,31 The additional sample dilution required by the DSC analysis might not fully reflect the protein properties in its actual formulation. Recently, Wang et al. demonstrated a microelectromechanical system (MEMS)-based DSC instrument.32 This novel technology measures heat using polydimethylsiloxane calorimetric chambers and nickel−chromium thermopiles. As shown by Wang et al., the sample material requirement is relatively low, ∼1.2 μL in volume, and the experimental setup seems to permit high protein concentrations, ∼1.4 mM lysozyme. According to the unique mechanism of detection, it is reasonable to assume that this DSC technique can be applied to much higher protein concentrations, presumably compatible with the requirement for mAb products. Furthermore, the microchannel composition will likely allow designs for higher throughput modules and, perhaps, automation-enabled devices. Though the results presented in this report seem to agree with the data found in the literature and acquired by traditional calorimetry methods,32 this new technique remains to be tested with more biopharmaceutics such as mAbs, considering that the two proteins examined, lysozyme and RNaseA, contain only single thermal transitions. In addition, the robustness and precision of the new DSC method need to be further demonstrated, and the compatibility with protein formulations needs to be tested, before it can be directly applied to the formulation development of protein therapeutics. Differential Scanning Fluorimetry. Differential scanning fluorimetry (DSF), otherwise known as thermal shift assay, was developed to characterize the shift in melting temperature of proteins and other macromolecular systems.33 This assay utilizes extrinsic fluorescent probes to monitor the thermal transition event exhibited by the sample. The hydrophobic dye, SYPRO Orange, was initially discovered as a protein stain (Invitrogen, Inc., Carlsbad, CA), and has been favored as the choice of an extrinsic fluorescent probe for DSF.34,35 This fluorescent molecule possesses great specificity and sensitivity to hydrophobic patches exposed by a protein during unfolding, as well as good compatibility with the thermocycler used for real-time polymerase chain reaction (RT PCR) detection.36 One typical application of DSF is during the screening for

large number of samples with low or no personnel attendance over a short period of time. Recently, additional signature features of high throughput technologies have been defined as low material and limited resource requirements. During the early development of pharmaceutical entities, especially before clinical evaluation, materials and resources are typically constrained. High throughput screening technology that provides a quick survey of product characteristics over a wide range of experimental conditions under reasonable cost will certainly add significant value to the success of clinical studies and commercial process and product development. With respect to mAb drug development, a commercial formulation is usually developed simultaneously as clinical trials are executed. Due to the high expense associated with making protein therapeutics, developers often do not commit to production of large quantities of material until positive clinical results are achieved. This makes high throughput methods particularly attractive to help guide the design and execution of formulation development. Moreover, the market demand for mAb products at higher protein concentrations is likely to bring more challenges, as the dosing restrictions are significantly higher for commercial products compared to those during clinical studies when formulation is still being developed. Over the past decade or so, high throughput technologies have been adopted for use in biophysical analyses and utilized in the early stages of protein formulation development. Many of these methods provide early characterization of protein physical properties such as solubility and viscosity, while biophysical measurements also serve as the primary analytical tools to detect protein product degradation including aggregation and precipitation under accelerated conditions. Furthermore, a number of high throughput techniques have been used to predict protein behavior in various formulations. The intention of this review is to present examples of the development of biophysical techniques with high throughput capability and their application in the formulation strategy of mAb products, rather than an exhaustive survey of the field.

■

HIGH THROUGHPUT BIOPHYSICAL TECHNOLOGY DEVELOPMENT AND APPLICATION A list of techniques discussed in this article is shown in Table 1, along with the corresponding applications and references. Differential Scanning Calorimetry. Modern differential scanning calorimetry (DSC) was first introduced in 1964.23 This technique provides unique characterization details regarding the thermal stability of individual protein domains. During the measurement, the heat required to maintain the same temperature increase between sample and reference cells is recorded as an indication of the heat capacity when the protein sample undergoes unfolding and other transitions that result in a heat difference.24 A typical DSC thermogram of a mAb contains three distinct transitions, belonging to the CH2, Fab and CH3 domains, respectively.5,22,25 It has been hypothesized that the thermal stability of a protein domain is correlated with its sensitivity to the environmental stress such as low pH and high temperature, which leads to protein unfolding and, subsequently, irreversible protein aggregation.22,26,27 Although it is unlikely that a protein therapeutic product will be subjected to temperatures higher than 40 °C, it is desirable to screen for formulation conditions where proteins are thermally stable. It is also important, prior to setting up real time formulation studies, to identify excipients that do not affect or improve protein thermal melting.28 It has been 698



Review

formulations. DSF has been compared closely to the traditional calorimetric method, DSC, and the results indicate that the melting temperatures obtained by the two techniques correlate nicely.11,40 As opposed to DSC, DSF consumes significantly less material and supports faster analysis. During protein formulation development, especially when large data sets are desired, DSF presents a more versatile tool to accommodate wide ranges of protein concentrations and formulations. Despite the many advantages that DSF offers, the method has a few limitations. Due to its hydrophobic nature, SYPRO Orange has a high binding affinity to organic solvents and detergents (He et al., unpublished results). Polysorbate molecules are commonly used as excipients in mAb formulations and other protein therapeutic products.42 DSF, however, does not allow analysis in the presence of polysorbates. Moreover, proteins during thermal melting can form irreversible aggregates as a result of molecules collapsing via unfolded parts and surfaces.22 This phenomenon is believed to be much enhanced at higher protein concentrations. It remains to be shown that DSF can differentiate the processes of unfolding and aggregation, especially at high protein concentrations.22 Additionally, DSF can only reveal the first thermal transition, as the detection of later ones is often compromised by the elevated fluorescence signal resulting from the early transition. Optical Density and Turbidity. Optical density measured in the visible light wavelength range, mostly 350−450 nm, is often referred to as turbidity. Since proteins usually do not contain internal components that absorb light specifically over this range, the light blockage is strictly related to the size of protein species. Turbidity measurements are commonly employed as an analytical method to detect changes in protein samples as a result of protein aggregation which leads to a size increase in protein species.43−45 Turbidity assessment can easily be integrated as part of a high throughput formulation screening strategy, by implementing multiwell based liquid handling protocols.45 It provides a direct readout of sample quality, especially upon accelerated stresses. In addition, turbidity is another technique that allows measurements at high protein concentrations, given that the baseline readings of unperturbed samples are low and one can avoid saturating the light detector by adjusting the path length. Many instruments also offer temperature control apparatus, which adds another option to further characterize protein aggregation behavior, as well as aggregation kinetics under different conditions. Turbidity measurements are especially useful in detecting the presence of large protein aggregates.43,46 Extensive protein aggregation that results in particulation poses a significant threat to the quality of protein therapeutics.7−9,47 Protein particulates often form upon mechanical stresses via contact with container surfaces or at liquid−air interfaces, and the particles typically fall into large, micrometer range, sizes (i.e., not resolvable by size exclusion chromatography).48−51 Since light blockage is proportional to the size of aggregates, turbidity measurement is suitable for particulation assessment in protein solutions. Many solution conditions have been shown to lead to extensive protein particulation, making it extremely important to identify formulations with low particulation propensity at early stages during product development. High throughput turbidity methods offer a resource-saving analysis sensitive to protein particulation when exploring a wide range of protein concentrations, excipients, and solution pH formulations, in

protein−ligand binding, where the association of the ligand and protein leads to a shift of the thermal transition to higher temperatures.37,38 Another utility of the method is for protein crystallography.37,39 The thermal stability detected by DSF is used as an indication of protein crystallization. Lately, the application of DSF has been highlighted in several reports.11−13,40,41 The high throughput format and low sample volume requirement (10−100 μL per sample) when using DSF in combination of multiwell RT PCR instruments offers great opportunities to analyze a variety of formulation conditions simultaneously. The hypothesized mechanism of detection is that, during protein unfolding, hydrophobic patches/sites are exposed and the extrinsic dye, SYPRO Orange, binds to these exposed hydrophobic areas, which results in the enhancement of fluorescence emission38 (Figure 2). The dye has low binding

Figure 2. Differential scanning fluorimetry (DSF). (A) Principle of DSF employing SYPRO Orange dye: protein unfolding caused by temperature increase leads to exposure of the hydrophobic patches that the fluorescent dye binds strongly, resulting in enhanced fluorescence emission. (B) Example of protein DSF melting curve: the midpoint of the first transition, denoted as Th or hydrophobic exposure temperature, is commonly used for sample comparison.

affinity to well folded proteins because the hydrophobic residues on proteins, such as mAbs, are typically buried on the interior in the folded state.36 This property allows SYPRO Orange to display greater selectivity to the unfolded conformation on a protein. In addition to the high throughput capability, DSF is compatible with high protein concentrations.40 Reliable results were obtained at mAb concentrations as high as 150 mg/mL (∼1 mM protein). Many hydrophobic fluorescent probes are pH sensitive and display significant fluorescence quenching under acidic conditions. SYPRO Orange, on the other hand, exhibits reasonable behavior from pH 4 to 8, the range where most protein therapeutics formulations are developed.40 The DSF procedure is also known to be programmable as most RT PCR instruments allow adjustment of key variables including heating rate, equilibration time, frequency of fluorescence reading, etc. One can optimize the screening methods or potentially develop short heating protocols to fully explore protein thermal stability in 699



Review

concentrations, as they often require a higher density of fluorophores. Additional changes resulting from molecule crowding as protein concentration increases might be detectable by these methods, but not achievable by steadystate measurements. Extrinsic Fluorescence Spectroscopy. Another commonly used technique is fluorescence spectroscopy employing extrinsic fluorophores. Hawe et al. published a thorough review article focused on the different types of extrinsic fluorescent probes that can be applied to protein characterization.59 Many of the available fluorescent dyes possess binding or interaction specificity to certain species of a protein population or specific domains within a protein. Another critical property of these dyes is that their fluorescence emission usually undergoes significant changes upon association or interaction with the target proteins. Among these dyes, hydrophobic probes including 1-anilinonaphthalene-8-sulfonate (ANS), 4,4′-bis-1anilinonaphthalene-8-sulfonate (bis-ANS), Nile Red, Congo Red and SYPRO Orange, are the most widely used in the characterization of protein therapeutics.59−62 These hydrophobic dyes are believed to probe the surface exposed hydrophobic regions on proteins, as a result of protein conformational changes. ANS and bis-ANS are probably the most characterized dyes of their class and, perhaps the most utilized ones in analyzing protein solution properties.59,63 They have been shown to have low background fluorescence signal when free in solution or bound to well-folded proteins, while expressing high affinity to the aggregated and structurally altered proteins with much enhanced fluorescence. The utility of SYPRO Orange in this application was inspired by the development of DSF.40,62 Similar to ANS and bis-ANS,63 SYPRO Orange was applied to the detection of temperatureinduced protein aggregates.62 It was shown that the detection method is sensitive only to the aggregated protein species that form at higher temperatures, but not to those formed during refrigerated long-term storage, indicating that the dye binding is directly related to the unfolded protein, not the size of aggregates.62 Another quickly emerging class of extrinsic fluorescent probes is the rotary dyes.64 Representatives of this class, such as 9-(dicyanovinyl)julolidine (DCVJ) and 9-(2carboxy-2-cyanovinyl)julolidine (CCVJ), are commonly used to reveal solvent polarity and viscosity, mostly in the absence of proteins.65 The method of detection relies on the difference in fluorescence emission and rotation frequency in response to different solvent properties.65 Recently, Hawe and co-workers discovered that CCVJ, besides its known application, can also be used to characterize protein aggregates.66 In addition, they were able to demonstrate that CCVJ fluorescence is not affected by polysorbates, which often results in high background signal in the presence of hydrophobic dyes. Much like intrinsic fluorescence spectroscopy, extrinsic fluorescence spectroscopy can be easily integrated into high throughput instrumentation as well. Many reports in the literature have demonstrated the feasibility of such methods in multiwell plate preparation and detection format.55,62,63,66 High throughput extrinsic fluorescence spectroscopy offers additional tools during the early development of protein formulations, due to the incapability of many analytical techniques in identifying protein species with physical degradations which can be detected by extrinsic fluorescent probes without further sample separation. The material and resource requirements of high throughput fluorescence spectroscopy, similar to other

conjunction with different physical stresses as well as types of containers. The disadvantage of the method, however, is also well understood.43 Though it offers an easy-to-use tool, it does not provide any information regarding protein species distribution or aggregate size. Furthermore, the measurement can be affected by the opalescent nature of high concentration protein solutions as often observed in mAb formulations.52 In order to apply multiwell plates when measuring turbidity, the turbidity detection mode requires the light to pass through the entire sample vertically, and might be significantly altered by the uneven settlement of large protein particles on the bottom of the sample well. Due to the above-mentioned limitations, high throughput turbidity assessment is often interpreted qualitatively rather than quantitatively. Intrinsic Fluorescence Spectroscopy. Fluorescence spectroscopy is one of the most developed techniques applied to characterize protein molecules both at the population level19,53 and at the single molecule level.54 Proteins are rich in internal fluorophores, such as the aromatic residues phenylalanine, tyrosine, and tryptophan. Steady-state fluorescence spectroscopy probing these intrinsic sites has been a useful tool to monitor changes in the microenvironment of such residues as an indication of protein tertiary conformation.20 As aromatic amino acid side chains become more buried, the quantum yield of fluorescence emission generally increases and the emission maximum shifts to lower wavelengths (blue shift). The opposite event takes place as the side chains become more solvent exposed. Therapeutic proteins are sensitive to many physical conditions that might lead to partial or complete unfolding of the protein. The unfolding changes are directly reflected by the changes in intrinsic fluorescence, as the method is often combined with elevated stress and long-term storage studies to probe global protein conformational stability.53,55 Microplate enabled fluorescence instruments have been used to increase the throughput capacity of fluorescence measurements, and to facilitate the noninvasive conformational assessment of protein therapeutics.55 With respect to mAbs, numerous aromatic amino acid side chains are scattered throughout the molecule, making it nearly impossible to allocate conformational changes detected by intrinsic fluorescence to a specific part of the mAb protein. Additional challenges exist as the strong fluorescence emission intensity of mAbs usually exceeds the detection limit of fluorometers even at moderate protein concentrations. Adjustments, such as decreasing the light path length, can be applied to accommodate the use at high protein concentrations.53 Overall, intrinsic fluorescence spectroscopy is a valuable technique that characterizes the global changes in protein tertiary conformation, and can be employed in a high throughput format during protein formulation studies. Besides the steady-state fluorescence measurements, other fluorescence techniques probing intrinsic fluorophores have also been successfully applied to the characterization of protein conformation in a high throughput format, including fluorescence anisotropy and fluorescence lifetime measurement.44,56,57 The application of these methods often relies on the distribution of the intrinsic fluorophores on proteins, and large protein molecules, such as mAbs, contain many aromatic side chains, which can cause significant challenges in the analysis and interpretation of the results. General information, however, can be obtained regarding protein interactions, as well as inter- and intramolecular dynamics.19,58 These techniques are potentially useful for protein characterization at high 700



Review

acceptable for proteins like mAbs which are large in monomeric size. Dynamic Light Scattering. Dynamic light scattering (DLS) is also known as quasi-elastic light scattering. It is an important tool to study macromolecular systems such as proteins, polymers and viruses, etc.74 Different from SLS, DLS measures the time-dependent fluctuation in light scattering intensity, caused by the Brownian motion of diffusants in solution.74 The pattern of intensity fluctuation of each scattering component is characterized by the autocorrelation function that is fitted to their decays. Information including size distribution of scattering centers, geometry, and diffusion patterns can be extrapolated from the autocorrelation function. Besides the size distribution, readouts from a DLS measurement also include the diffusion coefficient, through which the hydrodynamic radius can be calculated via the Stokes−Einstein equation. Currently, DLS is available in a high throughput format with microplate reader and temperature controller.75 DLS, particularly when applying intense laser source, provides an analytical range that typically covers from 1 nm to a few micrometers in radius, suitable for protein characterization. The main application of DLS in the development of protein therapeutics has been the detection of protein aggregates, including sub-micrometer protein particles.13,26,43,45,76 Large particles possess long decay times and contribute more significantly to the overall scattering intensity compared to small particles. This characteristic enables DLS to pick up signals arising from small populations of protein aggregates among highly concentrated monomeric proteins. High throughput DLS methods can be applied at different stages during the development of protein therapeutics and are particularly useful when comparing the protein particulation propensity in many formulations upon the physical stresses present during process and product distribution.77,78 Diffusion properties and hydrodynamic size measured by DLS have also been applied to characterize protein species in response to environmental changes such as pH and temperature.79,80 Another utility of DLS in protein characterization has emerged over the last decades, focusing on protein−protein interactions (PPI) as a result of electrostatic, van der Waals, hydrogen bonding and hydrophobic forces.81,82 Second virial coefficient, denoted as B22, is a thermodynamic parameter that reflects the nonideality of protein solutions at higher concentrations, depending on the geometry and interaction potentials of two interacting molecules in solution.83,84 Although static light scattering is often used to derive B22 values, the reduced form of B22 can be determined through the diffusion interaction parameter, kD, and the sedimentation interaction parameter, kS.72,85 Both interaction parameters have been shown to respond similarly to solution conditions such as ionic strength, pH and type of ions present. 85 The determination of kS requires time-consuming experiments using AUC and has significant limitation on protein concentration range that is suitable for analysis.85 The diffusion interaction parameter kD, on the other hand, can be empirically determined by measuring diffusion coefficient, D, as a function of diffusant (protein) concentration, using DLS.81,84,85 Because kD is a reflection of PPI and is sensitive to solution properties, it has been widely applied as a predictive tool to study protein aggregation propensity driven by or related to PPI, and to compare protein formulations.82,85 Besides aggregation propensity, protein viscoelastic behavior is also known to be revealed by kD.86−88 Since solution viscosity at high protein concen-

techniques implementing microplate technology, are very suitable to the early screening of protein formulation. Another potential application of extrinsic fluorescence is the detection of protein particulates. The hydrophobic dye, SYPRO Orange, was shown to strongly bind to protein particulates formed after agitation stress, and the fluorescence intensity appeared to correlate with the particle counts as determined by microflow imaging technique.62 In a later report, Mach and coworkers demonstrated a flow cytometry technique where SYPRO Orange was used to label protein particulates in the subvisible range (generally regarded as 1−125 μm).67 Another study published by Li et al. further implemented this method in a high throughput mAb formulation screening strategy.13 The high selectivity and sensitivity of SYPRO Orange enable the combination with flow cytometry and provide detailed information on the distribution as well as total amount of proteinaceous particulates, while light obscuration and microscopic methods often cannot differentiate proteins from environmental particles. Static Light Scattering. Static light scattering (SLS) is a technique that measures the amount of light scattered by the particles in solution.68,69 The scattered light is at the same wavelength as the source (usually monochromatic light), and the intensity of the scattered light is recorded and averaged over a short time period.68 Similar to turbidity assessment, SLS reflects the general information of the size of particles present in solution. SLS, when analyzed at multiple angles, can provide additional information on particle shape, as well as molecular mass.70 Scattering intensity is especially sensitive to the size of the particles and, therefore, can identify the presence of large protein species. SLS has been highly automated and applied to the study of protein formulations.11 The scattered light intensity can be measured by light scattering or fluorescence instruments coupled with high intensity laser and microplate accessories. The utility of this method in protein therapeutics development is mainly focused on the detection of protein aggregates formed after physical stresses such as accelerated temperatures and agitation.11,71 The high throughput application of SLS is extremely valuable for early formulation screening focused on protein aggregation and particulation propensity. It may also be used to screen for conditions that cause protein precipitation or to assess protein solubility. Since SLS is nondestructive, it offers good opportunities to follow the long-term kinetics of protein aggregation and precipitation, in order to differentiate among formulations. The amount of information obtained by SLS, however, is limited to the rough assessment of aggregation. Information regarding properties and distribution of protein aggregates requires further characterization. In addition, high concentration protein samples may lead to an inner-filter effect that can interfere with the measurement.69,72 SLS is often coupled with multiple detectors at different angles to the light source, and such a method is referred to as multiangle light scattering (MALS) or multiangle laser light scattering (MALLS) when a laser source is used. A MALS detector can be linked to fractionation instruments such as size exclusion chromatography (SEC) or field-flow fractionation (FFF) for online detection of fractionation protein species.61,73 The use of MALS synchronized with separation methods can help estimate the protein molecular mass and confirm the size and population of each aggregated species. While the accuracy of the mass determination is highly dependent on the instrument and solution properties, rough estimation is quite 701



Review

information on protein conformational properties, sample heterogeneity and distribution, identities of protein species, chemical modifications, and other critical aspects of protein therapeutic products. In recent years, the invention of ultraperformance liquid chromatography (UPLC) has been widely accepted in the field of biotechnology.98 UPLC applies smaller stationary particles compared to those used in HPLC, achieving expanded performance limits, such as pressure and mobile phase flow rate, as well as increasing separation efficiency and throughput. Analytical methods coupled with HPLC or UPLC have become an essential component of a high throughput strategy for mAb formulation screenings. Besides their main role as analytical tools to reveal protein degradation and sample quality, several new applications of liquid chromatography have been reported, with a specific focus on the characterization of mAbs. In an article published in 2007, Bajaj et al. presented an SEC method to study protein self-association propensity.99 Protein concentration and static light scattering are measured simultaneously as proteins elute and the second virial coefficient is determined. When the mobile phase is varied, proteins display different size distributions between dimers and monomers, and the protein’s propensity to dimerize can be obtained. It is hypothesized that dimerization often occurs during the initial step of extensive protein aggregation.100 This HPLC-based method presents a feasible tool to characterize protein self-association under various solution conditions, as a prediction of extensive protein aggregation. To this end, many researchers have demonstrated a more direct way to characterize protein interaction using liquid chromatography. One example widely used in protein characterization is selfinteraction chromatography.101 The technique measures protein elution from columns packed with resins conjugated to the same protein, and the second virial coefficient (B22) can be derived from the elution profile.102,103 More recently, this method was further improved by using commercially available polyclonal antibodies for column packaging instead of individual mAbs. Each mAb can be analyzed alone, and the elution time is dependent on the interactions between the mAb and the polyclonal antibodies present on the resin. Hence, the technique can be used to compare different mAbs based on the degree of interactions they display when passing through the same column. Jacobs et al. demonstrated that a panel of mAbs can be applied to the polyclonal antibody-containing columns and the results correlate with solubility properties of these proteins.104 The refined chromatographic method is particularly useful in the development of highly concentrated protein therapeutics. Potentially, different buffer conditions can also be coupled with the self-interaction chromatography in order to screen for formulations that allow the desired solubility. Other Techniques To Consider. Several other high throughput techniques have been extensively applied in pharmaceutical development, including UV spectroscopy, isothermal titration calorimetry (ITC), capillary electrophoresis (CE), etc. UV spectroscopy is the primary detection method for protein concentration.105 ITC is often applied to studying the ligand−protein interactions as part of target screening and drug design.106 CE and other electrophoresis methods are designed to characterize protein variants that differ by the ratio of net charges over mass, as an indication of sample impurity or degradation.95 Details regarding the applications of these techniques are not included in this article, as they have been thoroughly discussed elsewhere.

trations is believed to be strongly mediated by PPI and solution conditions that affect protein diffusion, DLS is a valuable technique to probe protein characteristics related to viscosity, a parameter that is critical to the product development of protein therapeutics.89 Furthermore, the high throughput capacity of DLS allows this type of characterization to be executed early in product development and, as a result, formulations without desired properties can be screened out prior to setting up large scale formulation studies. In an article published in 1991, de Smidt et al. demonstrated a DLS method to measure the viscosity of aqueous polymer solutions using latex spherical particles as a probe.90 A similar approach was reported and lysozyme was used as a diffusion probe to measure the viscosity of saline solutions containing different types of salts.91 In both cases, the solution viscosity was calculated from the diffusion coefficient, D, as detected by DLS, and following the Stokes−Einstein equation. A high throughput version of this technique was first applied to highly concentrated protein solutions in 2010.75 Due to the interference of protein scattering intensity, polystyrene beads were used as a probe manifesting sizes significantly greater than that of the protein (Figure 3). Additionally, the overall

Figure 3. Demonstration of the dynamic light scattering method for viscosity measurement. An example of the size distribution of the protein-bead mixture, measured by DLS, is illustrated on the right panel.

scattering intensity was dominated by the beads while the protein diffusion was affected by the nonideality at high concentrations, resulting in good separation between protein and bead species in size distributions.75 This technology was successively applied to the early screening of high concentration mAb formulations, where high throughput viscosity measurement was combined with high throughput thermal stability characterization.12 Information regarding formulation space, with desired protein properties, was determined by the statistical analysis of the high throughput screening results. The same method can also be used to characterize the effect of excipients on solution viscosity, providing further guidance for formulating mAb therapeutics at high concentrations.92 Liquid Chromatographic Methods. Liquid chromatographic methods are often used during the downstream process of protein therapeutics,93,94 as well as analytical measurements of protein samples.95 Commonly used chromatographic techniques in studying mAbs include SEC, ion-exchange chromatography (IEC), reversed phase (RP) chromatography, affinity chromatography, hydrophobic interaction chromatography (HIC), etc.96,97 The implementation of high performance liquid chromatography (HPLC) has enabled these methods to be employed at various scales with high efficiency and automation. The HPLC based analysis can provide detailed 702



■

Review

of most mAbs due to the increased Tm’s of many mAbs.11 While this is not an issue with most Peltier-controlled spectrophotometers, most plate readers that are commercially available are not capable of analysis at high temperatures, resulting in colloidal stability assessment and formulation screening to be conducted utilizing low throughput, cuvette based systems.53 In a recent study, Goldberg et al. demonstrated the use of the Stargazer 384 system which utilizes static light scattering in a 384-well format to assess mAb colloidal stability in a high throughput manner.11 The unique design of the Stargazer 384 system plate Peltier and optics systems allow samples to be subjected to temperatures upward of 85 °C. Through subjecting multiple IgG1s and IgG2s to thermal stress as a function of time, the stabilizing or destabilizing influence of various excipients and formulations on colloidal stability was demonstrated. This technology also has value for high throughput screening to assess colloidal stability of mAbs during the early research stages of molecule optimization and selection. A recent comprehensive study was conducted by Li et al. to assess the global aggregation behavior of two IgG2s and an IgG1 under different pH and ionic strength conditions utilizing multiple high throughput assays.13 In the case of this study, samples were incubated at high temperature and then transferred into multiwell plates for analysis. OD350 measurements were utilized to characterize aggregate size and the aggregate density, while DLS was employed to monitor changes in hydrodynamic radius and polydispersity. Recently, Zhao et al. illustrated the integration of a high throughput laboratory system that evaluates a large number of mAb formulations and incorporates numerous high throughput liquid handling techniques and multiwell plate-enabled analytical instruments. 45 In this formulation screening approach, different buffer and protein components are mixed by robotic systems and placed under storage, as well as accelerated temperature and agitation conditions, to assess protein stability in the respected formulations. The results are obtained using a number of high throughput techniques including automated lab-on-a-chip platform (ALP) and RPHPLC for impurity; SEC-HPLC, turbidity and DLS for aggregation and particulation; CEX-HPLC for protein charge variants.45 This highly automated system and analytical strategy demonstrate a great example of assessing protein quality and stability as part of high throughput mAb formulation screening. Characterization and Optimization of High Protein Concentration Solution Properties. Another critical factor of interest during liquid formulation development is the impact of various solutes, conditions, and excipients on the solubility of a mAb. Traditionally, assessment for solubility of proteins has proven to be difficult either due to a lack of quantity of the protein in question or due to physical changes in the protein at conditions near the solubility limit (formation of gel, precipitation, etc.). A recent study by Gibson et al. demonstrates the use of a high throughput polyethylene glycol (PEG)-induced solubility assay to assess the impact of varying pH and buffer species on the solubility of an IgG1.107 A solubility comparison of the same mAb construct expressed in different cell lines, as well as the same IgG1 in multiple formulations, was examined. Since the assay relies on a UV spectroscopic assessment of protein remaining in solution after PEG precipitation, the throughput of the traditional assay was greatly enhanced by utilization of an automated liquid handling system and a multiwell plate reader. This study provides an

UTILIZATION OF CURRENT HIGH THROUGHPUT TECHNOLGIES FOR FORMULATION SCREENING AND DEVELOPMENT As previously described, there are currently available high throughput technologies that have been demonstrated to be applicable to formulation screening and characterization of mAbs. For simplification, all technologies discussed below function by utilizing multiwell plate platforms, and describe screening/characterization assays which can be conducted in a rapid (several hours to a few days) manner relative to previous lower throughput (i.e., single sample analysis, etc.) technologies. In addition, the examples described below represent case studies that have been reported specifically for mAb development. Conformational Stability Screening. Assessment of the influence of various formulation factors on the conformational stability of a given mAb is usually conducted during early formulation development. The effectiveness of utilizing DSF as a high throughput conformational stability screening tool has been recently demonstrated on 4 different mAbs, of both the IgG1 and IgG2 isotypes.40 The high throughput nature of the DSF allowed 84 formulations to be screened to evaluate the influence of the various formulation adaptations on the conformational stability of the mAbs. To identify pH and buffer composition for optimal conformational stability, a pH and buffer species matrix was tested composed of twelve different conditions (acetate, phosphate, and histidine at pH’s within the buffering region of each buffer species). Depending on the number of replicates included for each sample, this type of screening can be conducted on one or two 96-well plates, significantly increasing the experimental throughput of the assay. The influence of individual excipients on the thermostability of these mAbs was also examined using DSF. The stabilizing and destabilizing effects observed by individual excipients correlated with previously collected DSC thermostability data. As previously discussed, the impact of protein concentrations on conformational stability screening was also demonstrated, indicating that thermostability rankings of various formulations could be reproduced at concentrations of up to 100 mg/mL. He et al. in a later study evaluated not only the use of DSF for the conformational stability screening of pH and excipients on a monoclonal antibody but also the impact of divalent cations on thermostability.12 A similar study by Goldberg et al. also demonstrated the use of DSF to screen the conformational stability of multiple formulations of several mAbs.11 As previously described, for most mAbs, the Tm or Th value reported by DSF correlates with the Tm of the CH2 domain unfolding, traditionally the least thermostable domain of an IgG as determined by DSC (Figure 3). Colloidal Stability Screening. The assessment of colloidal stability, the ability of particular formulations or other solution factors to inhibit aggregation, is of importance during protein formulation as it provides elucidation of potential formulation conditions that prevent protein aggregation. Traditional light scattering techniques have been employed to monitor protein aggregation for many years. One of the primary methods utilized is monitoring UV−vis absorbance at >350 nm, either in a single cuvette or in a higher throughput fashion using a multiwell plate reader. One of the major drawbacks for using this type of analysis method for monitoring and screening of aggregation of monoclonal antibodies is that higher temperatures (>65 °C) traditionally are required to induce aggregation 703



Review

accelerated studies. In many cases during early development, various forms of stresses which usually are intended to drive a given molecule away from its native or stable state are applied to identify the stabilizing or destabilizing influence of various formulation parameters (pH, excipients, buffer species, etc.). These stresses can range from applying increased temperatures in the form of thermal melts or increased temperature incubation, or in combination with stressing the molecule through changing pH and buffer species and placing a molecule in a “metastable” state.20 The application of the various stresses can have an impact on the ability of the data from the high throughput technologies to be predictive of actual product stability/characteristics. Often cases may arise where data from these various techniques will be in conflict with each other. For example, a particular excipient may be either conformationally of colloidally stabilizing in a particular mAb formulation, but may present issues in terms of increased viscosity or solubility issues of the product. Another example that could arise is that a formulation may be developed and optimized using high throughput techniques, and the given formulation is later discovered to rapidly form visible particles or cause large increases in subvisible particles, when placed on accelerated or real-time stability. Additional concerns surround the fact that most high throughput screening and characterization technologies utilize multiwell plates, which are not relevant container-closure systems in product filling. These types of screening and characterization studies do not represent potential formulation and stability issues associated with storage of a drug in glass vials or prefilled syringe in respect to potential interactions with silicone oil, tungsten, and various other extractable and leachable components.108 For the reasons discussed, the use of high throughput technologies during formulation development are usually limited to the early stages of product development to screen and limit the number of formulation conditions tested on stability, with most formulation development decisions and selections being made from accelerated or real-time stability data. As technology evolves, increasing numbers of techniques are becoming compatible with high throughput instrumentation. Future hardware and software advancements will bring more empirical tools to formulation screening strategy relevant to the actual storage behavior of mAbs in their final product presentation. More methodological development in high throughput biophysical technology is needed to increase the predictive power of protein properties that dictate the success of commercial products.

example of how a classical protein chemistry technique can be adapted to a much higher throughput screening assay through advancements in sample analysis and sample preparation technologies. During early formulation development, material limitations and sample requirements of traditional cone-and-plate rheometers make large scale formulation screening difficult. As previously mentioned, a high throughput DLS based viscosity screening technique for monoclonal antibodies has recently been published.75 This is primarily a result of the appearance of multiwell plate based DLS systems recently, allowing the adaptation of a previously existing technique developed for polymer solution viscosity assessments toward measuring the viscosity of high concentration mAb samples. The initial description of this technique by He et al. indicates that the DLS based method requires a 10-fold reduction in sample quantity over traditional cone-and-plate technologies, and can be conducted with a 5-fold reduction in time over manual viscosity measurements.75 Viscosity vs concentration curves were generated for both an IgG1 and an IgG2, over a concentration range from 0 to 140 mg/mL, indicating a good agreement with viscosity measurements obtained for the same molecules by traditional rheology techniques. In a follow-up study, He et al. demonstrated the increased throughput of this technology in a DOE study that modulated pH, divalent cation type, and excipients of an IgG2.12 In this particular study, the high throughput viscosity technique was utilized in parallel with the DSF assay for conformational stability. Another recent study utilizing this technique was aimed at elucidating effects of various sugar molecules and concentration on the viscosity of high concentration solutions of an IgG1 and an IgG2.92 This study demonstrated another increased throughput advantage of the DLS based assay during formulation screening and optimization, which is the ability to modulate sample temperature as needed, allowing viscosity measurements to be conducted at multiple temperatures using the plate of samples.

■

SUMMARY It is worth noting that while the use of high throughput techniques and strategies can be valuable in regard to saving time and utilizing small amounts of material, there are challenges to the implementation of the techniques as well. During formulation development of monoclonal antibodies, there are other stability factors that need to be monitored and assessed as a function of formulation conditions. These include but are not limited to fragmentation, chemical stability (oxidation and deamidation), subvisible particle assessments, bioactivity, and visual inspection for visible particulates. It should also be noted that while large numbers of high throughput technologies are feasible during early formulation development, it is critical that extensive caution be taken in the implementation of these assays during product development. Techniques have been discussed here that can characterize the impact of different formulation conditions (pH, excipients, buffer species, etc.) on various stability related attributes of a given mAb (conformational and colloidal stability, solubility, viscosity, etc.) in a high throughput manner. Utilizing the techniques in parallel during formulation development can become difficult if the predictive nature of the stability assessment obtained from each high throughput technique is not well understood in terms of how it translates to the actual stability profile of the molecule during either real-time or

■

AUTHOR INFORMATION

Corresponding Author

*H.S.S.: MedImmune, Department of Formulation Sciences, One MedImmune Way, Gaithersburg, MD 20878; tel, 301-3985436; fax, 301-398-7285; e-mail, [email protected]. F.H.: Amgen Inc., Drug Product Development, 1201 Amgen Court West, Seattle, WA 98119; tel, 206-265-6917; fax, 206217-0346; e-mail, [email protected].

■

ACKNOWLEDGMENTS

The authors are grateful to Drs. Hasige Sathish (MedImmune Inc.), Ambarish Shah (MedImmune Inc.), Steven Bishop (MedImmune Inc.), Ping Yeh (Amgen Inc.), Bruce Kerwin (Amgen Inc.) and Michael Treuheit (Amgen Inc.) for their 704



Review

(20) Maddux, N. R.; Joshi, S. B.; Volkin, D. B.; Ralston, J. P.; Middaugh, C. R. Multidimensional methods for the formulation of biopharmaceuticals and vaccines. J. Pharm. Sci. 2011, 100 (10), 4171− 97. (21) Welfle, K.; Misselwitz, R.; Hausdorf, G.; Hohne, W.; Welfle, H. Conformation, pH-induced conformational changes, and thermal unfolding of anti-p24 (HIV-1) monoclonal antibody CB4−1 and its Fab and Fc fragments. Biochim. Biophys. Acta 1999, 1431 (1), 120−31. (22) Vermeer, A. W.; Norde, W. The thermal stability of immunoglobulin: unfolding and aggregation of a multi-domain protein. Biophys. J. 2000, 78 (1), 394−404. (23) O’Neill, M. J. The Analysis of a Temperature-Controlled Scanning Calorimeter. Anal. Chem. 1964, 36 (7), 1238−45. (24) Freire, E. Differential scanning calorimetry. Methods Mol. Biol. 1995, 40, 191−218. (25) Chennamsetty, N.; Voynov, V.; Kayser, V.; Helk, B.; Trout, B. L. Design of therapeutic proteins with enhanced stability. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (29), 11937−42. (26) Ahrer, K.; Buchacher, A.; Iberer, G.; Jungbauer, A. Thermodynamic stability and formation of aggregates of human immunoglobulin G characterised by differential scanning calorimetry and dynamic light scattering. J. Biochem. Biophys. Methods 2006, 66 (1−3), 73−86. (27) Ishikawa, T.; Ito, T.; Endo, R.; Nakagawa, K.; Sawa, E.; Wakamatsu, K. Influence of pH on heat-induced aggregation and degradation of therapeutic monoclonal antibodies. Biol. Pharm. Bull. 2010, 33 (8), 1413−7. (28) Falconer, R. J.; Chan, C.; Hughes, K.; Munro, T. P. Stabilization of a monoclonal antibody during purification and formulation by addition of basic amino acid excipients. J. Chem. Technol. Biotechnol. 2011, 86 (7), 942−948. (29) Bhugra, C.; Pikal, M. J. Role of thermodynamic, molecular, and kinetic factors in crystallization from the amorphous state. J. Pharm. Sci. 2008, 97 (4), 1329−49. (30) Kawakami, K.; Pikal, M. J. Calorimetric investigation of the structural relaxation of amorphous materials: evaluating validity of the methodologies. J. Pharm. Sci. 2005, 94 (5), 948−65. (31) Treuheit, M. J.; Kosky, A. A.; Brems, D. N. Inverse relationship of protein concentration and aggregation. Pharm. Res. 2002, 19 (4), 511−6. (32) Wang, L.; Wang, B.; Lin, Q. Demonstration of MEMS-based differential scanning calorimetry for determining thermodynamic properties of biomolecules. Sens. Actuators, B 2008, 134 (2), 953−8. (33) Pantoliano, M. W.; Petrella, E. C.; Kwasnoski, J. D.; Lobanov, V. S.; Myslik, J.; Graf, E.; Carver, T.; Asel, E.; Springer, B. A.; Lane, P.; Salemme, F. R. High-density miniaturized thermal shift assays as a general strategy for drug discovery. J. Biomol. Screening 2001, 6 (6), 429−40. (34) Lo, M. C.; Aulabaugh, A.; Jin, G.; Cowling, R.; Bard, J.; Malamas, M.; Ellestad, G. Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery. Anal. Biochem. 2004, 332 (1), 153−9. (35) Malawski, G. A.; Hillig, R. C.; Monteclaro, F.; Eberspaecher, U.; Schmitz, A. A.; Crusius, K.; Huber, M.; Egner, U.; Donner, P.; MullerTiemann, B. Identifying protein construct variants with increased crystallization propensity--a case study. Protein Sci. 2006, 15 (12), 2718−28. (36) Lavinder, J. J.; Hari, S. B.; Sullivan, B. J.; Magliery, T. J. Highthroughput thermal scanning: a general, rapid dye-binding thermal shift screen for protein engineering. J. Am. Chem. Soc. 2009, 131 (11), 3794−5. (37) Vedadi, M.; Niesen, F. H.; Allali-Hassani, A.; Fedorov, O. Y.; Finerty, P. J. Jr.; Wasney, G. A.; Yeung, R.; Arrowsmith, C.; Ball, L. J.; Berglund, H.; Hui, R.; Marsden, B. D.; Nordlund, P.; Sundstrom, M.; Weigelt, J.; Edwards, A. M. Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (43), 15835−40.

critical review of the manuscript. The authors would also like to thank Dr. Jennifer Laurence for her assistance.

■

REFERENCES

(1) Dimitrov, D. S.; Marks, J. D. Therapeutic antibodies: current state and future trendsis a paradigm change coming soon? Methods Mol. Biol. 2009, 525, 1−27, xiii. (2) Reichert, J. M.; Rosensweig, C. J.; Faden, L. B.; Dewitz, M. C. Monoclonal antibody successes in the clinic. Nat. Biotechnol. 2005, 23 (9), 1073−8. (3) Wang, W.; Singh, S.; Zeng, D. L.; King, K.; Nema, S. Antibody structure, instability, and formulation. J. Pharm. Sci. 2007, 96 (1), 1− 26. (4) Furtado, P. B.; Whitty, P. W.; Robertson, A.; Eaton, J. T.; Almogren, A.; Kerr, M. A.; Woof, J. M.; Perkins, S. J. Solution structure determination of monomeric human IgA2 by X-ray and neutron scattering, analytical ultracentrifugation and constrained modelling: a comparison with monomeric human IgA1. J. Mol. Biol. 2004, 338 (5), 921−41. (5) Ionescu, R. M.; Vlasak, J.; Price, C.; Kirchmeier, M. Contribution of variable domains to the stability of humanized IgG1 monoclonal antibodies. J. Pharm. Sci. 2008, 97 (4), 1414−26. (6) Shire, S. J. Formulation and manufacturability of biologics. Curr. Opin. Biotechnol. 2009, 20 (6), 708−14. (7) Rosenberg, A. S. Effects of protein aggregates: an immunologic perspective. AAPS J. 2006, 8 (3), E501−7. (8) Carpenter, J. F.; Randolph, T. W.; Jiskoot, W.; Crommelin, D. J.; Middaugh, C. R.; Winter, G.; Fan, Y. X.; Kirshner, S.; Verthelyi, D.; Kozlowski, S.; Clouse, K. A.; Swann, P. G.; Rosenberg, A.; Cherney, B. Overlooking subvisible particles in therapeutic protein products: gaps that may compromise product quality. J. Pharm. Sci. 2009, 98 (4), 1201−5. (9) Singh, S. K. Impact of product-related factors on immunogenicity of biotherapeutics. J. Pharm. Sci. 2011, 100 (2), 354−87. (10) Manning, M. C.; Chou, D. K.; Murphy, B. M.; Payne, R. W.; Katayama, D. S. Stability of protein pharmaceuticals: an update. Pharm. Res. 2010, 27 (4), 544−75. (11) Goldberg, D. S.; Bishop, S. M.; Shah, A. U.; Sathish, H. A. Formulation development of therapeutic monoclonal antibodies using high-throughput fluorescence and static light scattering techniques: Role of conformational and colloidal stability. J. Pharm. Sci. 2011, 100 (4), 1306−15. (12) He, F.; Woods, C. E.; Trilisky, E.; Bower, K. M.; Litowski, J. R.; Kerwin, B. A.; Becker, G. W.; Narhi, L. O.; Razinkov, V. I. Screening of monoclonal antibody formulations based on high-throughput thermostability and viscosity measurements: Design of experiment and statistical analysis. J. Pharm. Sci. 2011, 100 (4), 1330−40. (13) Li, Y.; Mach, H.; Blue, J. T. High throughput formulation screening for global aggregation behaviors of three monoclonal antibodies. J. Pharm. Sci. 2011, 100 (6), 2120−35. (14) Pisal, D. S.; Kosloski, M. P.; Balu-Iyer, S. V. Delivery of therapeutic proteins. J. Pharm. Sci. 2010, 99 (6), 2557−75. (15) Shire, S. J.; Shahrokh, Z.; Liu, J. Challenges in the development of high protein concentration formulations. J. Pharm. Sci. 2004, 93 (6), 1390−402. (16) Kanai, S.; Liu, J.; Patapoff, T. W.; Shire, S. J. Reversible selfassociation of a concentrated monoclonal antibody solution mediated by Fab-Fab interaction that impacts solution viscosity. J. Pharm. Sci. 2008, 97 (10), 4219−27. (17) Kamerzell, T. J.; Middaugh, C. R. Two-dimensional correlation spectroscopy reveals coupled immunoglobulin regions of differential flexibility that influence stability. Biochemistry 2007, 46 (34), 9762−73. (18) Kamerzell, T. J.; Middaugh, C. R. The complex interrelationships between protein flexibility and stability. J. Pharm. Sci. 2008, 97 (9), 3494−517. (19) Kamerzell, T. J.; Ramsey, J. D.; Middaugh, C. R. Immunoglobulin dynamics, conformational fluctuations, and nonlinear elasticity and their effects on stability. J. Phys. Chem. B 2008, 112 (10), 3240−50. 705



Review

high throughput screening of serine/threonine kinases. Anal. Biochem. 2002, 308 (2), 223−31. (58) Ramsey, J. D.; Gill, M. L.; Kamerzell, T. J.; Price, E. S.; Joshi, S. B.; Bishop, S. M.; Oliver, C. N.; Middaugh, C. R. Using empirical phase diagrams to understand the role of intramolecular dynamics in immunoglobulin G stability. J. Pharm. Sci. 2009, 98 (7), 2432−47. (59) Hawe, A.; Sutter, M.; Jiskoot, W. Extrinsic fluorescent dyes as tools for protein characterization. Pharm. Res. 2008, 25 (7), 1487−99. (60) Dasnoy, S.; Dezutter, N.; Lemoine, D.; Le Bras, V.; Preat, V. High-throughput screening of excipients intended to prevent antigen aggregation at air-liquid interface. Pharm. Res. 2011, 28 (7), 1591− 605. (61) Hawe, A.; Friess, W.; Sutter, M.; Jiskoot, W. Online fluorescent dye detection method for the characterization of immunoglobulin G aggregation by size exclusion chromatography and asymmetrical flow field flow fractionation. Anal. Biochem. 2008, 378 (2), 115−22. (62) He, F.; Phan, D. H.; Hogan, S.; Bailey, R.; Becker, G. W.; Narhi, L. O.; Razinkov, V. I. Detection of IgG aggregation by a high throughput method based on extrinsic fluorescence. J. Pharm. Sci. 2010, 99 (6), 2598−608. (63) Hawe, A.; Rispens, T.; Herron, J. N.; Jiskoot, W. Probing bisANS binding sites of different affinity on aggregated IgG by steadystate fluorescence, time-resolved fluorescence and isothermal titration calorimetry. J. Pharm. Sci. 2011, 100 (4), 1294−305. (64) Kung, C. E.; Reed, J. K. Fluorescent molecular rotors: a new class of probes for tubulin structure and assembly. Biochemistry 1989, 28 (16), 6678−86. (65) Haidekker, M. A.; Brady, T. P.; Lichlyter, D.; Theodorakis, E. A. Effects of solvent polarity and solvent viscosity on the fluorescent properties of molecular rotors and related probes. Bioorg. Chem. 2005, 33 (6), 415−25. (66) Hawe, A.; Filipe, V.; Jiskoot, W. Fluorescent molecular rotors as dyes to characterize polysorbate-containing IgG formulations. Pharm. Res. 2010, 27 (2), 314−26. (67) Mach, H.; Bhambhani, A.; Meyer, B. K.; Burek, S.; Davis, H.; Blue, J. T.; Evans, R. K. The use of flow cytometry for the detection of subvisible particles in therapeutic protein formulations. J. Pharm. Sci. 2011, 100 (5), 1671−8. (68) Attri, A. K.; Minton, A. P. New methods for measuring macromolecular interactions in solution via static light scattering: basic methodology and application to nonassociating and self-associating proteins. Anal. Biochem. 2005, 337 (1), 103−10. (69) Minton, A. P.; Edelhoch, H. Light-Scattering of Bovine SerumAlbumin Solutions - Extension of the Hard Particle Model to Allow for Electrostatic Repulsion. Biopolymers 1982, 21 (2), 451−8. (70) Wang, T.; Lucey, J. A. Use of multi-angle laser light scattering and size-exclusion chromatography to characterize the molecular weight and types of aggregates present in commercial whey protein products. J. Dairy Sci. 2003, 86 (10), 3090−101. (71) Mahler, H. C.; Muller, R.; Friess, W.; Delille, A.; Matheus, S. Induction and analysis of aggregates in a liquid IgG1-antibody formulation. Eur. J. Pharm. Biopharm. 2005, 59 (3), 407−17. (72) Harding, S. E.; Johnson, P. The concentration-dependence of macromolecular parameters. Biochem. J. 1985, 231 (3), 543−7. (73) Li, Y.; Weiss, W. F. t.; Roberts, C. J. Characterization of highmolecular-weight nonnative aggregates and aggregation kinetics by size exclusion chromatography with inline multi-angle laser light scattering. J. Pharm. Sci. 2009, 98 (11), 3997−4016. (74) Schmitz, K. S. An introduction to dynamic light scattering by macromolecules; Academic Press: New York, 1990; 472 pp. (75) He, F.; Becker, G. W.; Litowski, J. R.; Narhi, L. O.; Brems, D. N.; Razinkov, V. I. High-throughput dynamic light scattering method for measuring viscosity of concentrated protein solutions. Anal. Biochem. 2010, 399 (1), 141−3. (76) Narhi, L. O.; Jiang, Y.; Cao, S.; Benedek, K.; Shnek, D. A critical review of analytical methods for subvisible and visible particles. Curr. Pharm. Biotechnol. 2009, 10 (4), 373−81. (77) Ahrer, K.; Buchacher, A.; Iberer, G.; Josic, D.; Jungbauer, A. Analysis of aggregates of human immunoglobulin G using size-

(38) Niesen, F. H.; Berglund, H.; Vedadi, M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat. Protoc. 2007, 2 (9), 2212−21. (39) Ericsson, U. B.; Hallberg, B. M.; Detitta, G. T.; Dekker, N.; Nordlund, P. Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal. Biochem. 2006, 357 (2), 289−98. (40) He, F.; Hogan, S.; Latypov, R. F.; Narhi, L. O.; Razinkov, V. I. High throughput thermostability screening of monoclonal antibody formulations. J. Pharm. Sci. 2010, 99 (4), 1707−20. (41) King, A. C.; Woods, M.; Liu, W.; Lu, Z.; Gill, D.; Krebs, M. R. High throughput measurement, correlation analysis and machine learning predictions for pH and thermal stabilities of Pfizer-generated antibodies. Protein Sci. 2011, 20 (9), 1546−57. (42) Kerwin, B. A. Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways. J. Pharm. Sci. 2008, 97 (8), 2924−35. (43) den Engelsman, J.; Garidel, P.; Smulders, R.; Koll, H.; Smith, B.; Bassarab, S.; Seidl, A.; Hainzl, O.; Jiskoot, W. Strategies for the assessment of protein aggregates in pharmaceutical biotech product development. Pharm. Res. 2011, 28 (4), 920−33. (44) Capelle, M. A.; Gurny, R.; Arvinte, T. High throughput screening of protein formulation stability: practical considerations. Eur. J. Pharm. Biopharm. 2007, 65 (2), 131−48. (45) Zhao, H.; Graf, O.; Milovic, N.; Luan, X.; Bluemel, M.; Smolny, M.; Forrer, K. Formulation development of antibodies using robotic system and high-throughput laboratory (HTL). J. Pharm. Sci. 2010, 99 (5), 2279−94. (46) Nayak, A.; Colandene, J.; Bradford, V.; Perkins, M. Characterization of subvisible particle formation during the filling pump operation of a monoclonal antibody solution. J. Pharm. Sci. 2011, 100 (10), 4198−204. (47) Joubert, M. K.; Luo, Q.; Nashed-Samuel, Y.; Wypych, J.; Narhi, L. O. Classification and characterization of therapeutic antibody aggregates. J. Biol. Chem. 2011, 286 (28), 25118−33. (48) Fesinmeyer, R. M.; Hogan, S.; Saluja, A.; Brych, S. R.; Kras, E.; Narhi, L. O.; Brems, D. N.; Gokarn, Y. R. Effect of ions on agitationand temperature-induced aggregation reactions of antibodies. Pharm. Res. 2009, 26 (4), 903−13. (49) Kiese, S.; Papppenberger, A.; Friess, W.; Mahler, H. C. Shaken, not stirred: mechanical stress testing of an IgG1 antibody. J. Pharm. Sci. 2008, 97 (10), 4347−66. (50) Bee, J. S.; Davis, M.; Freund, E.; Carpenter, J. F.; Randolph, T. W. Aggregation of a monoclonal antibody induced by adsorption to stainless steel. Biotechnol. Bioeng. 2010, 105 (1), 121−9. (51) Bee, J. S.; Stevenson, J. L.; Mehta, B.; Svitel, J.; Pollastrini, J.; Platz, R.; Freund, E.; Carpenter, J. F.; Randolph, T. W. Response of a concentrated monoclonal antibody formulation to high shear. Biotechnol. Bioeng. 2009, 103 (5), 936−43. (52) Salinas, B. A.; Sathish, H. A.; Bishop, S. M.; Harn, N.; Carpenter, J. F.; Randolph, T. W. Understanding and modulating opalescence and viscosity in a monoclonal antibody formulation. J. Pharm. Sci. 2010, 99 (1), 82−93. (53) Harn, N.; Allan, C.; Oliver, C.; Middaugh, C. R. Highly concentrated monoclonal antibody solutions: direct analysis of physical structure and thermal stability. J. Pharm. Sci. 2007, 96 (3), 532−46. (54) Weiss, S. Fluorescence spectroscopy of single biomolecules. Science 1999, 283 (5408), 1676−83. (55) Capelle, M. A.; Gurny, R.; Arvinte, T. A high throughput protein formulation platform: case study of salmon calcitonin. Pharm. Res. 2009, 26 (1), 118−28. (56) Owicki, J. C. Fluorescence polarization and anisotropy in high throughput screening: perspectives and primer. J. Biomol. Screening 2000, 5 (5), 297−306. (57) Fowler, A.; Swift, D.; Longman, E.; Acornley, A.; Hemsley, P.; Murray, D.; Unitt, J.; Dale, I.; Sullivan, E.; Coldwell, M. An evaluation of fluorescence polarization and lifetime discriminated polarization for 706



Review

exclusion chromatography, static and dynamic light scattering. J. Chromatogr., A 2003, 1009 (1−2), 89−96. (78) Ahrer, K.; Buchacher, A.; Iberer, G.; Jungbauer, A. Detection of aggregate formation during production of human immunoglobulin G by means of light scattering. J. Chromatogr., A 2004, 1043 (1), 41−6. (79) Brummitt, R. K.; Nesta, D. P.; Chang, L.; Chase, S. F.; Laue, T. M.; Roberts, C. J. Nonnative aggregation of an IgG1 antibody in acidic conditions: Part 1. Unfolding, colloidal interactions, and formation of high-molecular-weight aggregates. J. Pharm. Sci. 2011, 100 (6), 2087− 103. (80) Nobbmann, U.; Connah, M.; Fish, B.; Varley, P.; Gee, C.; Mulot, S.; Chen, J.; Zhou, L.; Lu, Y.; Shen, F.; Yi, J.; Harding, S. E. Dynamic light scattering as a relative tool for assessing the molecular integrity and stability of monoclonal antibodies. Biotechnol. Genet. Eng. Rev. 2007, 24, 117−28. (81) Chari, R.; Jerath, K.; Badkar, A. V.; Kalonia, D. S. Long- and short-range electrostatic interactions affect the rheology of highly concentrated antibody solutions. Pharm. Res. 2009, 26 (12), 2607−18. (82) Yadav, S.; Scherer, T. M.; Shire, S. J.; Kalonia, D. S. Use of dynamic light scattering to determine second virial coefficient in a semidilute concentration regime. Anal. Biochem. 2011, 411 (2), 292−6. (83) Saluja, A.; Kalonia, D. S. Nature and consequences of proteinprotein interactions in high protein concentration solutions. Int. J. Pharm. 2008, 358 (1−2), 1−15. (84) Li, S.; Xing, D.; Li, J. Dynamic Light Scattering Application to Study Protein Interactions in Electrolyte Solutions. J. Biol. Phys. 2004, 30 (4), 313−24. (85) Saluja, A.; Fesinmeyer, R. M.; Hogan, S.; Brems, D. N.; Gokarn, Y. R. Diffusion and sedimentation interaction parameters for measuring the second virial coefficient and their utility as predictors of protein aggregation. Biophys. J. 2010, 99 (8), 2657−65. (86) Yadav, S.; Shire, S. J.; Kalonia, D. S. Factors affecting the viscosity in high concentration solutions of different monoclonal antibodies. J. Pharm. Sci. 2010, 99 (12), 4812−29. (87) Yadav, S.; Shire, S. J.; Kalonia, D. S. Viscosity analysis of high concentration bovine serum albumin aqueous solutions. Pharm. Res. 2011, 28 (8), 1973−83. (88) Yadav, S.; Sreedhara, A.; Kanai, S.; Liu, J.; Lien, S.; Lowman, H.; Kalonia, D. S.; Shire, S. J. Establishing a link between amino acid sequences and self-associating and viscoelastic behavior of two closely related monoclonal antibodies. Pharm. Res. 2011, 28 (7), 1750−64. (89) Yadav, S.; Liu, J.; Shire, S. J.; Kalonia, D. S. Specific interactions in high concentration antibody solutions resulting in high viscosity. J. Pharm. Sci. 2010, 99 (3), 1152−68. (90) de Smidt, J. H.; Crommelin, D. J. A. Viscosity measurement in aqueous polymer solutions by dynamic light scattering. Int. J. Pharm. 1991, 77 (2−3), 261−4. (91) Parmar, A. S.; Muschol, M. Lysozyme as diffusion tracer for measuring aqueous solution viscosity. J. Colloid Interface Sci. 2009, 339 (1), 243−8. (92) He, F.; Woods, C. E.; Litowski, J. R.; Roschen, L. A.; Gadgil, H. S.; Razinkov, V. I.; Kerwin, B. A. Effect of sugar molecules on the viscosity of high concentration monoclonal antibody solutions. Pharm. Res. 2011, 28 (7), 1552−60. (93) Shukla, A. A.; Hubbard, B.; Tressel, T.; Guhan, S.; Low, D. Downstream processing of monoclonal antibodiesapplication of platform approaches. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2007, 848 (1), 28−39. (94) Andrew, S. M.; Titus, J. A. Purification of immunoglobulin G. Curr. Protoc. Immunol. 2001, Chapter 2, Unit 2 7. (95) Ahrer, K.; Jungbauer, A. Chromatographic and electrophoretic characterization of protein variants. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2006, 841 (1−2), 110−22. (96) Flatman, S.; Alam, I.; Gerard, J.; Mussa, N. Process analytics for purification of monoclonal antibodies. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2007, 848 (1), 79−87. (97) Bakshi, M.; Singh, S. Development of validated stabilityindicating assay methodscritical review. J. Pharm. Biomed. Anal. 2002, 28 (6), 1011−40.

(98) Toyo’oka, T. Determination methods for biologically active compounds by ultra-performance liquid chromatography coupled with mass spectrometry: application to the analyses of pharmaceuticals, foods, plants, environments, metabonomics, and metabolomics. J. Chromatogr. Sci. 2008, 46 (3), 233−47. (99) Bajaj, H.; Sharma, V. K.; Kalonia, D. S. A high-throughput method for detection of protein self-association and second virial coefficient using size-exclusion chromatography through simultaneous measurement of concentration and scattered light intensity. Pharm. Res. 2007, 24 (11), 2071−83. (100) Mahler, H. C.; Friess, W.; Grauschopf, U.; Kiese, S. Protein aggregation: pathways, induction factors and analysis. J. Pharm. Sci. 2009, 98 (9), 2909−34. (101) Tessier, P. M.; Vandrey, S. D.; Berger, B. W.; Pazhianur, R.; Sandler, S. I.; Lenhoff, A. M. Self-interaction chromatography: a novel screening method for rational protein crystallization. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2002, 58, (Partt 10 No. 1), 1531−5. (102) Tessier, P. M.; Lenhoff, A. M.; Sandler, S. I. Rapid measurement of protein osmotic second virial coefficients by selfinteraction chromatography. Biophys. J. 2002, 82 (3), 1620−31. (103) Tessier, P. M.; Sandler, S. I.; Lenhoff, A. M. Direct measurement of protein osmotic second virial cross coefficients by cross-interaction chromatography. Protein Sci. 2004, 13 (5), 1379−90. (104) Jacobs, S. A.; Wu, S. J.; Feng, Y.; Bethea, D.; O’Neil, K. T. Cross-interaction chromatography: a rapid method to identify highly soluble monoclonal antibody candidates. Pharm. Res. 2010, 27 (1), 65−71. (105) Mach, H.; Arvinte, T. Addressing new analytical challenges in protein formulation development. Eur. J. Pharm. Biopharm. 2011, 78 (2), 196−207. (106) Ui, M.; Tsumoto, K. An approach to rational ligand-design based on a thermodynamic analysis. Recent Pat. Biotechnol. 2010, 4 (3), 183−8. (107) Gibson, T. J.; McCarty, K.; McFadyen, I. J.; Cash, E.; Dalmonte, P.; Hinds, K. D.; Dinerman, A. A.; Alvarez, J. C.; Volkin, D. B. Application of a high-throughput screening procedure with PEGinduced precipitation to compare relative protein solubility during formulation development with IgG1 monoclonal antibodies. J. Pharm. Sci. 2011, 100 (3), 1009−21. (108) Bee, J. S.; Randolph, T. W.; Carpenter, J. F.; Bishop, S. M.; Dimitrova, M. N. Effects of surfaces and leachables on the stability of biopharmaceuticals. J. Pharm. Sci. 2011, 100 (10), 4158−70.

707


Advancements in High Throughput Biophysical Technologies

Recommend Documents