If therapeutic proteins are to be safe and efficacious, their misfolding and aggregation behaviors must be well understood.1 These behaviors are usually clarified through detailed structural characterization, particularly of the secondary structure of the protein, using established technologies such as conventional Fourier transform infrared (FTIR) spectroscopy and far-ultraviolet circular dichroism (far-UV CD). However, these technologies may clash with workflow requirements.2
An alternative technology is microfluidic modulation spectroscopy (MMS). It is a next-generation technique that optimally shapes IR spectroscopy for biopharmaceutical development. MMS, then, can measure previously undetectable changes in protein structural attributes, changes that are critical to drug efficacy and quality.3
Proteins typically misfold or aggregate in response to thermal stress or in the presence of chaotropic agents, molecules that reduce structural order by disrupting noncovalent bonds. The resulting structural change tends to be irreversible, which is suboptimal when it comes to studying the kinetics and thermodynamics of aggregation; reversible change enables more detailed investigation. Reversible misfolding and aggregate dissociation can be promoted by the application of high hydrostatic pressure, which makes pressure cycling technology (PCT) a valuable, orthogonal tool for aggregation studies.4
This tutorial showcases the combined application of MMS and PCT to quantify aggregation behavior and includes experimental data from a study of the aggregation of human immunoglobin G (IgG). IgG is routinely used as a model system for monoclonal antibodies (mAbs), the largest class of biopharmaceutical target molecules, so the data demonstrate the wide applicability of this powerful combination of technologies.
Changing protein structure through the application of pressure
The application of hydrostatic pressures of around 100 MPa and above modifies protein conformation, even in the absence of chemical or thermal energy, by forcing water molecules into hydrophobic pockets within the protein structure. This leads to structural disruption and the dissociation of macromolecular complexes and aggregates. Such changes are protein-specific, highly reproducible—unlike entropic thermal denaturation—and generally reversible.
PCT, as exemplified by the Barocycler 2320EXT (Pressure BioSciences), enables the cycling of pressure (up to 58,000 psi/400 MPa), facilitating studies that are highly complementary to those involving thermal denaturation or chaotropic agents. The partnering of PCT with a suitably sensitive characterization technique is essential to effectively detect and quantify induced structural changes.
Changes in the primary motifs of secondary structure, such as β-sheet and α-helix levels, are directly associated with misfolding and aggregation. Far-UV CD is an incumbent technology for the measurement of secondary structure, but it is best suited to dilute, simple solutions in which the protein represents the only chromophores; buffer choice is also restricted. These limitations are important when it comes to assessing the stability of complex, concentrated, commercially representative formulations.
In addition, FTIR is a commonly used spectroscopic technique that quantifies secondary structure via probing of the amide band. Absorption features across the band are well correlated with secondary structure.5 Indeed, IR is particularly sensitive to the changes in intermolecular β-sheet structure that are indicative of aggregate formation.6 That said, conventional FTIR technology suffers from widely recognized practical limitations. Its autosampler functionality is minimal, and its concentration range does not extend far below 10 mg/mL. Another common issue is background drift, which can compromise sensitivity.
MMS is a new technology that measures the secondary structure of proteins in complex samples across a sample concentration range from 0.1 to 200 mg/mL. It incorporates
- a high-power, quantum cascade laser that can generate an optical beam around 1000 times brighter than those used for conventional FTIR.
- a microfluidic flow cell to rapidly modulate the sample with a matched background buffer and produce differential absorption spectra.
- fully automated sample handling.
These features combine to deliver high-resolution, drift-free absorption spectra across the amide I band, and these spectra may be converted into detailed structural information via the robust correlations established from conventional FTIR.
Using PCT and MMS to study the aggregation of Human IgG1
Experimental details: In a case study, samples of human IgG1 with an initial concentration of ~4.5 mg/mL were prepared in phosphate-buffered saline (PBS) and n-propanol (10%) and then exposed to elevated pressure for 15 minutes (Barocycler 2320EXT) before being returned to ambient conditions. Tests were carried out at 40, 50, 60, and 90 Kpsi, at ambient temperature with a control conducted at atmospheric pressure.
A further sample was heated to 80°C for two minutes, to compare the effects of thermal stress, and then allowed to cool. Samples were subsequently centrifuged (15,000 × g for 10 minutes), and the resulting supernatant liquid was subject to a Bradford assay to determine protein concentration.
All samples were measured at ambient temperature by MMS (AQS3pro, RedShiftBio) using a microfluidic transmission cell of approximately 23.8-µm pathlength; modulation frequency was 1 Hz; and a backpressure of 5 psi was applied. Test conditions were automatically optimized by the instrument software (AQS3delta). Data were acquired at 33 discrete wavenumbers across the amide I band from 1714 cm−1 to 1590 cm−1 with replicate measurements carried out for each sample. Data analysis was carried out using the proprietary software for the instrument.
Results: Results for the PBS samples are shown in Figure 1. The opacity of the samples remains constant up to a treatment pressure of 60 Kpsi, and concentration measurements confirm that it is only treatment at 90 Kpsi that causes a significant reduction in protein concentration of protein in the supernatant liquor. This reduction is attributable to aggregation and precipitation at the highest treatment pressure.
Higher order structure estimates for the samples confirm minimal change as a result of treatment at 40 and 50 Kpsi or at elevated temperature. However, at 60 Kpsi, around 6.5–7.0% of the parallel β-sheet content had converted to an antiparallel β-sheet structure. This is good evidence of the initial stages of aggregation.
The results for the samples containing 10% n-propanol are very different, highlighting the effect of the aliphatic co-solvent. Treatment at either 60 kPSI or 80°C results in samples that are clearly opaque, and concentration data (see Figure 1) confirm that both conditions are associated with significantly reduced protein concentrations in the supernatant liquor. These data indicate a synergistic effect between the co-solvent and pressure that results in irreversible aggregation and precipitation.
MMS data show progressive change in the structure of the protein at increasing pressure. These changes are highlighted by the presentation of second-derivative data (see Figure 2). Estimates of higher order structure show the progressive and proportionate conversion of parallel to antiparallel β-sheet content and an increase in α-helical regions at the expense of β-turns, an observation entirely consistent with published literature. It is worth noting that the high turbidity of some of these samples had no impact on measurement sensitivity.
The presented data highlight the potential to stimulate misfolding and aggregation through the application of pressure and to sensitively detect the resulting change using the novel technique of MMS. The application of high hydrostatic pressure to study aggregation is a relatively new concept, but there is a growing literature to support its use along with commercial technology to facilitate the application of this orthogonal technique. MMS has an important role to play in all aggregation studies by sensitively detecting structural change with high resolution. Full automation adds to its appeal and enables the rigorous investigation of misfolding and aggregation in a streamlined and highly efficient workflow.
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