Produced in Partnership with RedShiftBioReviewed by Maria OsipovaOct 10 2024
Biologic drugs are naturally large and complex, making it virtually impossible to create accurate replicas of them as generic drugs. If companies can prove that their products are very similar to a biologic that has already received US Food and Drug Administration approval, they can be labeled as “biosimilar.”
Image Credit: RedShiftBio
Trastuzumab (Herceptin®) is a drug used for certain types of cancer, specifically for patients who are HER2-positive.1 Many manufacturers make biosimilars of this drug for research and clinical purposes. For a drug to qualify as a biosimilar, it should first pass rigorous characterization that shows identical properties to the originator.2
The following research examined the originator’s structure and two research-grade biosimilars via microfluidic modulation spectroscopy (MMS), a new, superior tool for structural characterization. All three samples were further assessed under regular fluctuation conditions in phosphate-buffered saline (PBS) and after 30 minutes of 70 °C elevated temperature to examine their stability.
MMS probes the amide I band of the IR spectrum to carefully examine protein structures while modulating against the reference buffer for precise, real-time background subtraction in water-based samples. Owing to its sensitivity, MMS is especially useful for quality control and works with various formulation buffers.
The second-generation MMS system, Apollo, was used in this study. It is fitted with a high-power quantum cascade laser that is considerably more intense than traditional Fourier transform infrared spectroscopy (FTIR) sources of light.
MMS is around 30-fold more sensitive than FTIR and five-fold more sensitive than CD, owing to small variations in its structure. This stems from a blend of more light and modulation of background subtraction.3
Methods
Trastuzumab (Herceptin®) was received as a dry powder with the salts of its formulation parts and in its original packaging. The pre-portioned powder was re-suspended with 7.4 mL high-performance liquid chromatography water to increase the protein and buffer to the appropriate concentrations.
A stock of corresponding formulation buffer was generated from 18.4 mg/mL trehalose, 0.459 mg/mL L-histidine HCl monohydrate, 0.297 mg/mL L-histidine, and 0.081 mg/mL PS20. The two biosimilars were already dissolved in formulation buffer or PBS.
The samples were then split into two aliquots each. One was dialyzed in formulation buffer, while the other was dialyzed in PBS to ensure each sample was accompanied by an optimally matching reference buffer to properly assess its effects on their structure and stability.
As the biosimilars were provided at 5 mg/mL and the trastuzumab was prepared at 21 mg/mL, the neat trastuzumab, alongside a 5 mg/mL diluted sample, was set in motion to contrast the concentration effects.
All samples were run three times on an AQS3pro system fitted with a flow cell with a pathlength of 22.3 µm and sweep-scanning abilities. The modulation speed was 1 Hz, and the backing pressure was 5 psi.
The trastuzumab samples in each of the buffers were placed in a 70 °C water bath for 30 minutes and then assessed in MMS after cooling to room temperature. This was carried out to identify structural change and examine the buffer’s impact on the effects of temperature stress. All results are displayed as the three replicates’ averages.
Results
Comparing the Originator to Two Biosimilars
Neat trastuzumab (21 mg/mL), diluted trastuzumab (5 mg/mL), and the two biosimilars (5 mg/mL) were all tested in the formulation buffer.
Figure 1 demonstrates the MMS results—among them, the absolute spectra (A), the second derivative (SD) spectra (B), the similarity plot (C), and the HOS fractional contribution profile (D). Absolute spectra were normalized for concentration and buffer contribution. Meanwhile, the second derivative plot displays areas of small variation.
By inverting and baseline subtracting the second derivative plot, the similarity plot was generated and was ultimately fit with Gaussian curves to calculate the fractional content of the higher order structure (HOS).
Table 1. A quantitative analysis of concentration, repeatability, and comparison to Trastuzumab innovator. Source: RedShiftBio
Figure 1. Absolute absorbance (A), second derivative (B), similarity (C), and HOS bar charts are shown above for the originator Trastuzumab at 5 and 21 mg/mL and two biosimilars each at 5 mg/mL. All spectra are well overlaid and appear as just one trace, but are actually four traces. The HOS bar chart highlights the similarities in structure.
The colors correspond to: Trastuzumab 5 mg/mL is red, Trastuzumab 21 mg/mL is purple, biosimilar 1 is green, and biosimilar 2 is blue. Image Credit: RedShiftBio
Formulation Buffer vs. PBS
All samples were dialyzed in the formulation buffer and PBS to compare the buffer’s effect on antibody structure. Figure 2 displays the second derivative (A), HOS bar graph (B), and the delta plot (C) for each of the samples in the formulation buffer at 5 mg/mL concentration and in PSB.
Table 2. Quantitative measurements of area of overlap showing repeatability among replicates and similarity compared to the Trastuzumab in formulation buffer are all highly similar. Source: RedShiftBio
The delta plot is a visual approach for comparing samples by subtracting the SD of each sample from a control. The dashed lines constitute the replicate variation. Traces above or below the dashed lines signal changes in spectra.
In this study, no samples demonstrated any changes linked to the buffer. Table 2 quantitatively demonstrates that all samples were comparable, as none of the similarities differed by over 1 % compared to the control.
Figure 2. Second derivative plot (A), HOS bar chart (B), and delta plot (C) show each sample in PBS is highly similar to the samples in formulation buffer.
The lighter shades are in FB and the darker shades are in PBS. The Trastuzumab samples are red, biosimilar 1 is green, and biosimilar 2 is blue. Image Credit: RedShiftBio
Heat Stress Qualitative Results
Trastuzumab in PBS and formulation buffer was heated to 70 °C for 30 minutes before being cooled to room temperature and running on MMS. Figure 3 shows the second derivative (A) and delta plot (B).
The two heated samples demonstrated heightened spectra (around 1624 cm-1) and a reduction in the area (at 1640 cm-1, native β-sheet). The results signify a small structural variation for the sample in the formulation buffer and larger variations for the sample in PBS. This indicates that trastuzumab has more resistance to temperature stress in the formulation buffer than in PBS.
Figure 3. Second derivative plot (A) of Trastuzumab in FB at room temp (light blue) and after heat stress (bright red), and Trastuzumab in PBS at room temp (dark blue) and after heat stress (dark red).
(B) shows the delta plot and highlights regions of change, and (C) and (D) are the delta plots separating out the samples in FB and in PBS, respectively. Image Credit: RedShiftBio
Heat Stress Quantitative Results
Figure 4A displays the HOS bar graph for each of the samples. While heat stress did not lead to major structural changes, the figure suggests a reduction in β-sheet content and a rise in turn structure due to heat stress. The weighted spectral difference (WSD) was quantified and is shown in Figure 4C as a graph, where the error bars signify ± 2x the standard deviation among replicates.
The WSD and the percent similarities both show that the formulation buffer heated sample underwent subtle structural variation and that the heated PBS sample demonstrated a more significant structural variation.
This may mean that trastuzumab undergoes an initial denaturing pathway when the stress temperature nears its melting temperature (Tm1 = 71 °C), where some β-sheets start to unfold and transform into β-turn structures.
Table 3. Area of overlap calculated for the repeatability for each sample and comparison to the RT control in FB. Source: RedShiftBio
This β-sheet to turn transition propensity is even more visible in the sample in PBS than in formulation, emphasizing the significance of formulation buffer in maintaining antibody stability.
Figure 4. HOS bar charts for Trastuzumab in FB at room temp (light blue), heat-treated (bright red), and in PBS at room temp (dark blue), and heat-treated (dark red). Section (B) shows a more detailed view of the beta-sheet and turn structure changes and the trend that temperature stress has on Tras.
The weighted spectral difference is shown in (C) and the change is clearly visible for both samples, however, the sample in PBS shows a much larger spectral change. Image Credit: RedShiftBio
Conclusions
Trastuzumab and the two biosimilars had exceedingly similar secondary structures at both formulation concentrations and when diluted to 5 mg/mL, sustaining an AO exceeding 99 % compared to the control. PBS had no effect on antibody structure, as calculated via the delta plot and AO similarities when compared to the samples in the formulation buffer.
Heat stress can affect the structure of trastuzumab, and the sample in PBS was more severely affected than that in formulation. Heat stress led to a loss in β-sheet content and a gain in turn structure, which may suggest the unraveling of the native structure.
MMS proved a robust, automated instrument that can be quickly and easily used to examine protein structure for biosimilar comparisons and identify variation in a quantitative (via the percent similarity, HOS percentages, or WSD) and qualitative (via the delta plots) manner.
MMS can streamline the decision-making process when assessing samples for comparability, and enhanced biophysical characterization instruments like MMS will facilitate the production of superior biosimilar drugs.
References and Further Reading
- Vulto, A. and Jaquez, O. (2017) The process defines the product: what really matters in biosimilar design and production? Rheumatology, 56(4), pp.14-29.
- Spector, N. and Blackwell, K. (2009) Understanding the mechanisms behind trastuzumab therapy for human epidermal growth factor receptor 2–positive breast cancer. Journal of Clinical Oncology, 27(34), pp.5838-5847.
- Brent, K. et al. (2020) Determining spectroscopic quantitation limits for misfolded structures." Journal of Pharmaceutical Sciences, 109(1), pp.933-936.
About RedShiftBio
RedShiftBio is redefining the possibilities for analyzing protein structure and concentration.
RedShiftBio has developed a proprietary life sciences platform combining our Microfluidic Modulation Spectroscopy (MMS) and expertise in high-powered quantum cascade lasers that provide ultra-sensitive and ultra-precise measurements of molecular structure. These structural changes affect critical quality attributes governing the safety, efficacy, and stability of biomolecules and their raw materials. This combination of technologies is available to researchers in our fully-automated Aurora and Apollo systems and is backed by a global network of sales, applications, service, and support teams to address all market needs.
Alongside our commitment to further innovation in the formulations and development space, RedShiftBio also supports biopharmaceutical manufacturing with HaLCon, our bioprocess analytics platform, purpose-built to measure protein titer at time of need.
Led by an experienced management team with a proven track record of success in both large instrumentation companies and commercializing disruptive technologies, RedShiftBio is here to support your research, development, and manufacturing goals. Our instruments can be found in the majority of the leading biopharmaceutical companies and CDMOs in the world. We also run product demonstrations and process samples in the StructIR Lab, located in our Boxborough, MA headquarters, as well as at partner sites including the Wood Centre in Oxford, UK, Spectralys/UCB in Brussels, Belgium, and at Sciex laboratories in Redwood Shores, CA.
RedShiftBio is backed by Waters Corporation, Illumina Ventures, Technology Venture Partners, and one undisclosed leading life science company.
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