Book Approaches To The Conformational Analysis Of Biopharmaceuticals Protein Science
Generating a stable environment for a biopharmaceutical drug substance is a critical step for ensuring a long drug-product shelf life 1—6. This process begins early in development with preformulation screening. Some of the most critical parameters to maintaining potency and activity are protein conformation tertiary or three-dimensional 3-D structure , folding secondary structure , and proper subunit association quaternary structure. Collectively, those are known as higher-order structure HOS and can be highly influenced by the formulation environment of a protein drug product.
But analytical monitoring of protein conformation is frequently omitted from preformulation because of time constraints, the complexity of most common techniques, and the inherent lack of sensitivity of some biophysical methods to subtle structural changes. However, combining traditional biophysical techniques with more sensitive orthogonal approaches can provide a comprehensive view of HOS in biopharmaceutical formulation. Biophysical analysis has become increasingly important in the characterization of biopharmaceutical drug candidates.
Such methods may be described as covering a mixture of disciplines, being essentially the use of physical techniques to investigate and characterize biological systems 7. The wide range of technologies within this area for elucidating 3-D protein structure includes spectroscopic, thermodynamic, and hydrodynamic techniques such as circular dichroism CD 8—11 , Fourier-transform infrared FTIR 12—15 spectroscopy, differential scanning calorimetry DSC , intrinsic and extrinsic fluorescence 16, 17 , dynamic light scattering DLS , and sedimentation velocity analytical ultracentrifugation SV-AUC 18— Such analyses can provide valuable information about secondary, tertiary, and quaternary structures of protein molecules e.
Biophysical analyses have particular application in determining and confirming the stability and conformation of protein structures.
They also can be used to define optimum buffer conditions and characterize degradation pathways and products. So biophysical analysis provides valuable information about how a formulation environment influences stability of a protein molecule 21— These techniques also provide information that can be used for comparability analysis.
The Role of Biophysical Analysis in Product Development The emergence and rapid growth of the biosimilars market is driving growing integration of biophysical analyses into biomolecular characterization 22, About 15 years ago, the biopharmaceutical industry was somewhat skeptical about biophysical analysis. Although it was considered to be state-of-the-art technology, widespread acceptance of biophysical methods was hindered by a lack of compliance and streamlined purpose to fulfill regulatory and practical requirements of the biopharmaceutical industry e. Consequently, its use remained underdeveloped in the biopharmaceutical sector.
The emergence of biosimilars changed that status quo. Biosimilar drug manufacturers needing to provide technical comparability data found biophysical analysis to be a valuable analytical tool. Visionary contract research organizations CROs such as Avacta Analytical and SGS M-Scan were early promoters of the use of this type of analysis, but they faced a challenge in persuading customers of its true value. Generally, larger companies with extensive testing capabilities already were fully committed to using such techniques for protein characterization. But most medium-to-small biopharmaceutical companies have neither the capacity nor the internal expertise to implement biophysical analysis.
Unfortunately, the need for HOS characterization often was widely ignored at many levels. Funds needed to invest in the required technology were neither available nor justifiable by those companies. Furthermore, regulatory guidelines ICH Topic Q6B did not sufficiently emphasize the relevance of biophysical analysis — nor do they today — and challenges associated with collection and productive interpretation of the so-called spectroscopic profiles. In addition to that ominous picture, lack of a competitive CRO market providing biophysical services perpetuated that vicious cycle until the arrival of biosimilars.
Together with technological advances in instrumentation, market education on HOS was part of this recurring issue. Over recent years, that less-than-bright picture has improved substantially, with gradual inclusion of biophysical instrumentation as part of routine analyses used for biopharmaceutical characterization. As a response to a greater presence of CROs active in this area, such instrumentation has become more streamlined to current industry requirements.
Meanwhile, regulatory guidelines are becoming more proactive in emphasizing the importance of protein HOS analysis. Perhaps more important, an increasingly demanding market and more educated industry is the force behind these deterministic changes. Both can be insensitive to subtle tertiary conformational changes that may be caused by small differences in the formulation environment. Below are data obtained from analyzing three different formulations of bovine immunoglobulin G IgG1 in an attempt to reduce the electrostatic dimeric content of IgG.
SV-AUC data indicate that one formulation successfully mitigated oligomerization of bovine IgG but induced small changes in its thermal stability and surface hydrophobicity area. By contrast, formulations B and C at t 0 showed less dimerization Figure 1, Table 1. SV-AUC measurements indicated that both B and C were successful in mitigating the electrostatic interactions that led to formation of dimeric species.
Extrinsic fluorescence measurements confirmed that view. Figures 3 and 4 show a small but different linear response in the fluorescence emission spectra of bis-ANS as a function of concentration of IgG in formulation C relative to formulation A and B. CD Figures 5 and 6, Table 3 , FTIR Figure 7, Table 4 , and intrinsic fluorescence measurements Figure 8 aiming to elucidate the secondary and tertiary structure of proteins showed that the different composition of formulations A, B, and C did not affect the structure of bovine IgG.
Table 1: Relative abundance of monomers to dimers of IgG in three replicate formulations A, B, and C are determined by calculating the integrated areas of the c s distributions. Those data showed a loss of ellipticity in the far UV from t Changes in tertiary structure were identified at t 15 in near UV. Significant changes in the fluorescence spectra of bovine IgG in formulation C were detected as early as t 15 days. From t 0 to t 45, a progressive increase in fluorescence emission quantum yield and blue shift of the emission spectra was observed Figure Conclusions: Commonly used FTIR and CD methods for determining protein structural conformation can be insensitive to subtle tertiary conformational changes induced by small changes in the formulation environment.
Data obtained from analyzing three different formulations of bovine IgG1 showed that formulation C was successful in mitigating the oligomerization of bovine IgG at the expense of inducing small changes in its thermal stability and surface hydrophobicity area. The minor differences detected on thermal stability and exposure of hydrophobic residues on the surface of bovine IgG are understood to be induced by subtle changes in the ensemble tertiary structure of IgG as a response to its formulation in buffer C.
Our orthogonal use of biophysical methods prompted a more detailed investigation of the refrigerated stability capabilities of bovine IgG in formulation C. Resulting refrigerated stability data showed that bovine IgG formulated in that buffer system had a propensity to lose tertiary and secondary structure along set time points that were not seen with formulations A and B data not shown.
Those results confirmed our view that formulation C was a successful buffer system to reduce electrostatic dimerization of bovine IgG but that it induced subtle tertiary structural changes at t 0. Those changes, by exposure of an increased hydrophobic surface area, were critical to the refrigerated conformational stability of bovine IgG in that specific formulation. These data demonstrate the importance of selecting orthogonal stability-indicating methods for conformational stability in formulation studies.
Key biophysical markers of stability can be overviewed easily or omitted otherwise. Minor conformational changes can expose regions of a protein molecule that are normally protected within its internal structure, potentially leading to unexpected degradation. Either way, the aim is always to optimize a strategy in relation to the intended purpose of the study.
The difficulty lies in that all biophysical methods have their limitations, so no single technique provides all required information. In many cases, the analysis of HDX MS data not only allows the loss of conformational integrity within a pharmaceutical product to be easily detected and localised, but also the mechanism of ensuing loss of activity to be understood. The MS-based methods of characterising protein conformation had been applied successfully in recent years to probe various aspects of higher order structure of large protein drugs; nevertheless, some challenges still remain.
Perhaps the most formidable challenge is presented by the heterogeneity of many biopharmaceutical products for example, due to extensive glycosylation or conjugation to a synthetic polymer. Ultimately, the success of MS-based tools as a means to probe conformation of recombinant therapeutic proteins will be determined by their adaptability to the specific needs of the biopharmaceutical industry. For example, reproducibility both between laboratories and across the platforms of these measurements is a critical issue that still needs to be addressed.
Although a significant amount of work will be required in the near future in order to address this and other questions, these efforts are well justified, since adoption of MS for the new role in the biopharmaceutical sector highlighted in this article will become a boon to the analytical characterisation and quality control. Furthermore, availability of these new tools will provide fresh impetus in other areas as well, for example by catalysing development of new proteinbased therapies.
The award recognizes DIA community chairs for consistently driving engagement while advancing knowledge and thought leadership within their membership communities. The current pharmaceutical market has faced a variety of challenges, including increasing expectations for quality from end-users and regulatory agencies driven by concern for patient safety. While pharmaceutical companies are working to assure that new quality and compliance paradigms are met, a balance must be achieved between the reality of managing costs in an effort to provide a product that meets the requirements of payers along with facilitating profitability in order to continue adequate business reinvestment.
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Download PDF file. Send to a friend. Download Adobe Reader. European Biopharmaceutical Review Protein Production. Misfolding Proteins Failure to fold or maintain the native conformation has a negative impact on the efficacy of the protein drug, since the ability to interact with physiological targets requires that the native conformation be maintained throughout the lifecycle of a protein molecule.
Mass Spectrometry-based Tools Despite being a relatively recent addition to the biopharmaceutical analysis toolkit, biological MS has already become a default method for characterising the covalent structure of protein therapeutics 1,2. Electrospray Ionisation Mass Spectrometry Two MS-based techniques show particular promise as a means of probing conformational properties of protein therapeutics. Conclusion The MS-based methods of characterising protein conformation had been applied successfully in recent years to probe various aspects of higher order structure of large protein drugs; nevertheless, some challenges still remain.
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