Conjugate vaccines are developed by linking molecules that are poorly immunogenic to a carrier protein to improve immunogenicity. They can be highly effective, as exemplified by the Haemophilus Influenzae b (Hib) and Prevnar 13® (which prevents infections from pneumococcal bacteria) vaccines, both of which are conjugates. However, conjugate vaccines are complex to formulate and costly, with very few carrier proteins licensed for use.
FinaBio (Fina Biosolutions LLC, Rockville, MD), a developer of conjugate vaccines, has developed an E. coli expression system to produce CRM197, one of the most commonly used carrier proteins, to improve the accessibility of this important class of vaccines.1 In this article we consider the analytical strategies applied to demonstrate the biophysical and immunological comparability of the new carrier protein, and to optimize its formulation.
Introducing a New Carrier Protein for Conjugate Vaccines
CRM197 is a genetically detoxified diphtheria toxin with several characteristics that can prove challenging for formulators and manufacturers. A relatively labile protein, it is prone to nicking and degradation by multiple routes including dimerization, aggregation, and precipitation. Notably it is pH sensitive with a tendency to turn inside out under low (acidic) pH conditions and can dimerize via domain exchange. Formulation conditions are controlled to keep conformational change and degradation to a minimum with comprehensive biophysical characterization applied to ensure that the CRM197 remains structurally intact under the conditions of use, up to the point of patient delivery.
Historically, CRM197 has been expressed in Corynebacterium diphtheriae but yields are generally low. More recently CRM197 has been expressed using periplasmic expression in Pseudomonas fluorescence and E. coli.2 Intracellular expression of CRM197 in E. coli has been challenging as it forms inclusion bodies, likely due to the bacteria’s reducing cytoplasm which causes disulfide-bonded proteins like CRM197 to form inclusion bodies.3
FinaBio has developed an E. coli strain with an oxidative environment and used it to express intracellular CRM197. This strain, which is not crippled and grows to very high densities, expresses a properly folded, soluble protein which is then purified at 2g/L via two ion exchange chromatography steps. Marketed as EcoCRM®, this CRM197 has the same primary sequence as CRM197 and can be considered akin to a biosimilar or generic form of the reference protein. However, as production involves cytoplasmic expression in a new host as opposed to secreted/periplasmic and the purification process is also new, demonstrating biophysical and immunological equivalence is essential.
Demonstrating Biophysical and Immunological Comparability
In an initial series of biophysical studies, the higher order structure of EcoCRM® was compared with that of CRM197 from Corynebacterium and from Pseudomonas.4 Proteins with identical primary structure can exhibit different therapeutic performance on account of their secondary, tertiary or quaternary structure and therefore be rigorously compared to confirm comparability.
Observation and conservation of a protein’s secondary structural components, such as alpha-helix and beta-sheet is critical to the demonstration of bioequivalence. The tertiary structure of a protein is its overall three-dimensional shape which is primarily defined by non-covalent bonding between the R groups of the constituent amino acids. However, disulfide bonds also have an important impact, not least because of their significant strength relative to other contributing bonds. As already discussed, these bonds are particularly relevant to the functionality of CRM197. In the preliminary studies the secondary structure of the CRM197 samples was characterized by circular dichroism (CD), a widely used technique that is most effective for simple, dilute samples and tertiary structure was characterized using three different techniques: second derivative UV analysis; intrinsic fluorescence; and extrinsic fluorescence (which involves the use of an added fluorophore). All these early analyses supported a claim of comparability in the EcoCRM® and provided preliminary evidence that it is correctly folded.
Satisfactorily confirming biosimilarity can be particularly challenging for biotherapeutic proteins and it is common to apply a battery of orthogonal biophysical tests to robustly identify any detectable difference. In further work, an additional analytical technique, Microfluidic Modulation Spectroscopy, was therefore applied to extend the evidence base for comparability between the secondary structure of the EcoCRM® and one of the existing commercial sources of CRM197.
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MMS is a technique that generates drift-free, background compensated differential infra-red (IR) absorption scans of the Amide I band by modulating the sample with a matching buffer through the detector during sample acquisition. This band is associated with the C=O stretch vibration of peptide linkages along the protein backbone and is sensitive to changes in secondary structure.
Figure 1 shows the percent composition of secondary structure determined by MMS analyses of the two sources of CRM197. These data complement the previously acquired results, improving the totality of evidence of similarity by directly comparing the composition of secondary structural elements and demonstrating the high conservation of secondary structure. The percentage of beta-turn, alpha helix, unordered and beta-sheet structure in each type of CRM197 is closely similar and well-matched to levels observed via crystal structure analysis of the diphtheria toxin.
Working Towards an Optimized Formulation
As a result of successfully applying MMS to provide evidence of similarity in secondary structure the FinaBio team decided to incorporate the technique in formulation studies. Unlike CD, MMS measures consistently and reliably across a wide range of concentrations, and in the presence of excipients, a defining feature of the technology that brings considerable value in formulation studies. The preceding results also highlight it as the more informative technique for the characterization of secondary structure, with the capability to more precisely quantify secondary structural elements.5
As part of the development work, stability trials were carried out with two different formulations, made up with ‘Old’ and ‘New’ buffers. Samples were held for up to 12 weeks at two temperatures (20°C and 4°C) with analysis carried out after one, two, four and twelve weeks. A freshly thawed sample was used for reference.
Figure 2 shows the MMS data gathered over the course of the stability trial. Replicates for each sample of the new formulation are very consistent demonstrating the high repeatability of the technique once the effect of concentration, which varies slightly across the sample set, is taken into account. Absolute spectra (Figure 2a), corrected for the effect of concentration, overlay very closely indicating highly consistent secondary structure under all storage conditions.
Second derivative plots, which help to highlight similarity or difference in a set of samples, were produced for both formulations using the instrument data analysis software (AQS3delta - Figure 2b). These data clearly indicate differences with respect to stability over the 12-week period. Area of Overlap analysis, a numerical analysis of the extent to which the area under one second derivative curve overlaps with that of another, was carried out to quantify this difference and was used to produce plots of dissimilarity (Figure 2c). These plots show that in the old buffer the CRM197 exhibits differences in structural change after 1 week at room temperature (orange bars) and after 2 weeks at 4°C (gold bars). In contrast, in the new buffer, structural change was not observed until the 12-week time point. In the new formulation the protein is only around 1% dissimilar from the control after 12 weeks at room temperature (dark blue bars).
The spectral scans were further processed to directly compare the amount of each element of sub-structure in the stored new formulation and freshly thawed control samples. This analysis confirmed that the measured elements of secondary structure – betasheet, beta-turn, alpha-helix and unordered – were preserved in the new formulation for the duration of the trial, with variability across each individual element typically no more than 1 – 2%, across all the samples (data not shown).
These results indicate that the new buffer improves formulation stability, a result consistent with other analyses carried out as part of the formulation study. For example, sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS PAGE) also indicates that in the new buffer the EcoCRM® remains intact after 12 weeks at room temperature. SDS PAGE analysis under reducing conditions results in the appearance of some faint lower molecular weight bands, indicating a limited amount of nicking, but the protein remains within specification. Size Exclusion Chromatography (SEC) shows no evidence of dimer or aggregate formation in the new formulation, at either temperature.
In combination the analyses point to the superior performance of the new buffer which was subsequently selected for formulation of the GMP product that the company is progressing to commercialization.
Conclusion
Demonstrating biosimilarity or comparability in a new biotherapeutic protein is a challenging task and calls for the application of a battery of biophysical characterization techniques. This example study illustrates the value of MMS in such studies and for formulation optimization. By generating highly repeatable, detailed information about secondary structure it provides robust confirmation of comparability. The ability to measure across a wide range of concentrations and in the presence of excipients is particularly useful when it comes to formulation studies.
References
- Broker, M.; Costantino, P.; DeTora, L.; McIntosh, E. D.; Rappuoli, R., Biochemical and biological characteristics of cross-reacting material 197 CRM197, a non-toxic mutant of diphtheria toxin: use as a conjugation protein in vaccines and other potential clinical applications. Biologicals 2011, 39 (4), 195-204.
- Stefan, A.; Conti, M.; Rubble, D.; Ravagli, L.; Presta, E.; Hochkoeppler, A., Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli. Journal of biotechnology 2011, 156 (4), 245-52.
- Goffin, P.; Dewerchin, M.; De Rop, P.; Blais, N.; Dehottay, P., High-yield production of recombinant CRM197, a non-toxic mutant of diphtheria toxin, in the periplasm of Escherichia coli. Biotechnology journal 2017, 12 (7).
- Hickey et al. ‘Analytical Comparability Assessments of 5 Recombinant CRM197 Proteins’ J Pharm Sci 107:1806, 2018
- Kendrick, B. S. et al. (2019) ‘Determining Spectroscopic Quantitation Limits for Misfolded Structures’, Journal of Pharmaceutical Sciences. Elsevier Ltd, 109(1), pp. 933–936.