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NMR Higher order structure (HOS) analysis of biologics for comparison studies of batch-to-batch consistency, biosimilars and formulation optimizations

Salam Al-Karadaghi
Salam Al-Karadaghi

Unlike generic small-molecule drugs, which are synthesized in the laboratory and are exact copies of the reference drug, monoclonal antibodies are produced in mammalian cells due to the ability of these cells to perform post-translational modification. A drawback of expression in heterologous systems is unpredictable modifications like variable glycosylation, deamination of asparagine and glutamine, and oxidation of cysteine, methionine, or tryptophan, which result in heterogeneity in the biologic product. Unexpected modifications may affect the product's chemical properties, such as surface charge, solubility, stability, and three-dimensional structure (higher order structure or HOS). These, in turn, may affect the efficacy and safety of the treatment. For this reason, biosimilars cannot be handled as generic small-molecule drugs (and they are not considered "generic drugs"). For approval by regulatory agencies, developers must present detailed comparative studies of the biosimilar, which has to be highly similar to the reference antibody, with no clinical differences in safety, quality, and efficacy, as stated by EMA. Often, methods like UV and fluorescent spectroscopy are used for tertiary structure analysis, while Fourier Transform Infrared (FTIR) and CD spectroscopy are used for secondary structure analysis. However, X-ray crystallography and NMR spectroscopy yield much more accurate and detailed structural information. NMR spectroscopy in a time- and cost-efficient manner can be used to analyze higher order structure (HOS) of intact antibodies or other biologics. For example, a powerful application is to accurately and efficiently compare a biosimilar's higher order structure (HOS) to its originator antibody or compare production lots to ensure higher order structure batch-to-batch consistency. At SARomics Biostructures, we routinely use 2D-NMR to compare the HOS of biosimilars to originator biologics or as a verification method for the HOS of different production batches. Spectra can easily be analyzed by visual inspection and extended by different analysis methods such as peak analysis, ECHOS, or chemometric methods such as principal component analysis (PCA). The poster shown here was presented at the Protein and Antibody Engineering Summit (PEGS Europe) in Lisbon (November 14-16, 2023).

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NMR Higher order structure (HOS) analysis of biologics for comparison studies of batch-to-batch consistency, biosimilars and formulation optimizations Authors: Olof Stenstr旦m, Carl Diehl and Bj旦rn Walse SARomics Biostructures AB, Lund, Sweden www.saromics.com Natural abundance NMR as a tool for HOS comparison of biosimilars and batch control NMR spectroscopy can be used to analyze higher order structure (HOS) of intact antibodies or other biologics. For example, a powerful application is to accurately and efficiently compare a biosimilars higher order structure to its originator anti- body (Figure 1) or compare production lots to ensure higher order structure batch-to-batch consistency. At SARomics Biostructures we routinely use 2D-NMR as a method to compare the HOS of biosimilars to originator biologics or as a verification method for HOS of different production batches. Spectra can easily be analysed by visual inspection and can be extended by different analysis methods such as peak analysis, ECHOS or using chemometric methods such as principal component analysis (PCA) (see Figure 1 and Figure 2). Contact: Carl Diehl, NMR Services Manager, Senior Scientist, carl.diehl@saromics.com Olof Stenstr旦m, Scientist, olof.stenstrom@saromics.com Forced oxidation of infliximab studied using time resolved 2D natural abundance NMR spectroscopy Intact or cleaved mAbs can be studied and characterized using 2D-NMR. It should be noted that cleavage is not necessary for 2D-NMR analysis, especially if one wishes to do HOS studies of the mAb in formulation buffer. In a joint pro- ject, SARomics Biostructures together with Genovis AB (Lund, Sweden) used FabALACTICA速 to cleave originator and biosimilar versions of infliximab followed by characterization and comparison using 2D-NMR. As part of this project, forced oxidation using sodium peroxide was performed, where the oxidization of the methionines could be detected without changing the overall higher order structure of the cleaved Fab/Fc domains. In addition, forced oxidation of the Fc domain were studied by time-resolved 2D-NMR with non-uniform sampling (NUS) to reduce spacing between spectra (see Figure 3 and Figure 4). Figure 3: 1 H-13 C 2D NMR spectra of inflix- imab as native Fab/ Fc (orange) and af- ter forced oxidation using sodium peroxide (blue). Box indicate region containing methionine side cha- ins. Box indicate region containing methionine side chains. Figure 5: 1 H-13 C 2D NMR spectra of native hGH (orange) and un- folded hGH at a SDS/hGH ratio of 37 (green). Figure 6: Zoom in on the methionine region of 1 H-13 C 2D NMR spectra of native hGH (orange), hGH at SDS/hGH ratio of 4 (blue), hGH at a SDS/hGH ratio of 37 (green) and unfolded hGH at a SDS/hGH ratio of 360 (red). Figure 4: Time- resolved oxidation process of infliximab Fc followed using 1 H-13 C 2D NMR spectra at 0 (red), 2 (green) and 16 h (blue). Top panels show zoom in on the methionine re- gion (A) and oxidized methionine region (B). Excipient effect on HOS studied using 2D natural abundance NMR Natural abundance 2D-NMR can be used to characterize changes to HOS due to the influence of various excipients. SARomics Biostructures is a member of Next- BioForm, an academic-industrial consortium focused on the formulation of bio- logical molecules, where SARomics Biostructures contributes its expertise in NMR spectroscopy and structural biology. As part of this consortium, the effect of SDS on human growth hormone (hGH) was studied using a variety of physical characterization techniques including 2D-NMR and SANS. The data clearly show how the secondary and tertiary structure unfolds with increasing SDS concen- tration. As part of this project, the effect of various excipients on the formulation of biologics are studied using a toolbox of different techniques, where 2D-NMR serves as a vital characterization technique for higher order structure (see Figure 5 and Figure 6). Reference: Sanchez-Fernandez,Adrian, Carl Diehl, Judith E. Houston,Anna E. Leung, James P.Tellam, Sarah E. Rogers, Sylvain Prevost, Stefan Ulvenlund, Helen Sj旦gren, and Marie Wahlgren. An integrative toolbox to unlock the structure and dynamics of proteinsurfactant complexes. Nanoscale Advances 2, no. 9 (2020): 4011-4023. Figure 2: 1 H-13 C 2D NMR spectra of NIST Fab pro- duced in 20%-13 C-labeled P. pastoris (orange) or murine (green) expression media. Boxes indicate where differences for methyl groups can be detected in the spectra. www.saromics.com Figure 1: ECHOS analysis of 2D-NMR spectra for quantification of similarity between originator and biosimilar. Box indicate how specific peak differences can be identified from the ECHOS analysis.

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NMR Higher order structure (HOS) analysis of biologics for comparison studies of batch-to-batch consistency, biosimilars and formulation optimizations

  • 1. NMR Higher order structure (HOS) analysis of biologics for comparison studies of batch-to-batch consistency, biosimilars and formulation optimizations Authors: Olof Stenstr旦m, Carl Diehl and Bj旦rn Walse SARomics Biostructures AB, Lund, Sweden www.saromics.com Natural abundance NMR as a tool for HOS comparison of biosimilars and batch control NMR spectroscopy can be used to analyze higher order structure (HOS) of intact antibodies or other biologics. For example, a powerful application is to accurately and efficiently compare a biosimilars higher order structure to its originator anti- body (Figure 1) or compare production lots to ensure higher order structure batch-to-batch consistency. At SARomics Biostructures we routinely use 2D-NMR as a method to compare the HOS of biosimilars to originator biologics or as a verification method for HOS of different production batches. Spectra can easily be analysed by visual inspection and can be extended by different analysis methods such as peak analysis, ECHOS or using chemometric methods such as principal component analysis (PCA) (see Figure 1 and Figure 2). Contact: Carl Diehl, NMR Services Manager, Senior Scientist, carl.diehl@saromics.com Olof Stenstr旦m, Scientist, olof.stenstrom@saromics.com Forced oxidation of infliximab studied using time resolved 2D natural abundance NMR spectroscopy Intact or cleaved mAbs can be studied and characterized using 2D-NMR. It should be noted that cleavage is not necessary for 2D-NMR analysis, especially if one wishes to do HOS studies of the mAb in formulation buffer. In a joint pro- ject, SARomics Biostructures together with Genovis AB (Lund, Sweden) used FabALACTICA速 to cleave originator and biosimilar versions of infliximab followed by characterization and comparison using 2D-NMR. As part of this project, forced oxidation using sodium peroxide was performed, where the oxidization of the methionines could be detected without changing the overall higher order structure of the cleaved Fab/Fc domains. In addition, forced oxidation of the Fc domain were studied by time-resolved 2D-NMR with non-uniform sampling (NUS) to reduce spacing between spectra (see Figure 3 and Figure 4). Figure 3: 1 H-13 C 2D NMR spectra of inflix- imab as native Fab/ Fc (orange) and af- ter forced oxidation using sodium peroxide (blue). Box indicate region containing methionine side cha- ins. Box indicate region containing methionine side chains. Figure 5: 1 H-13 C 2D NMR spectra of native hGH (orange) and un- folded hGH at a SDS/hGH ratio of 37 (green). Figure 6: Zoom in on the methionine region of 1 H-13 C 2D NMR spectra of native hGH (orange), hGH at SDS/hGH ratio of 4 (blue), hGH at a SDS/hGH ratio of 37 (green) and unfolded hGH at a SDS/hGH ratio of 360 (red). Figure 4: Time- resolved oxidation process of infliximab Fc followed using 1 H-13 C 2D NMR spectra at 0 (red), 2 (green) and 16 h (blue). Top panels show zoom in on the methionine re- gion (A) and oxidized methionine region (B). Excipient effect on HOS studied using 2D natural abundance NMR Natural abundance 2D-NMR can be used to characterize changes to HOS due to the influence of various excipients. SARomics Biostructures is a member of Next- BioForm, an academic-industrial consortium focused on the formulation of bio- logical molecules, where SARomics Biostructures contributes its expertise in NMR spectroscopy and structural biology. As part of this consortium, the effect of SDS on human growth hormone (hGH) was studied using a variety of physical characterization techniques including 2D-NMR and SANS. The data clearly show how the secondary and tertiary structure unfolds with increasing SDS concen- tration. As part of this project, the effect of various excipients on the formulation of biologics are studied using a toolbox of different techniques, where 2D-NMR serves as a vital characterization technique for higher order structure (see Figure 5 and Figure 6). Reference: Sanchez-Fernandez,Adrian, Carl Diehl, Judith E. Houston,Anna E. Leung, James P.Tellam, Sarah E. Rogers, Sylvain Prevost, Stefan Ulvenlund, Helen Sj旦gren, and Marie Wahlgren. An integrative toolbox to unlock the structure and dynamics of proteinsurfactant complexes. Nanoscale Advances 2, no. 9 (2020): 4011-4023. Figure 2: 1 H-13 C 2D NMR spectra of NIST Fab pro- duced in 20%-13 C-labeled P. pastoris (orange) or murine (green) expression media. Boxes indicate where differences for methyl groups can be detected in the spectra. www.saromics.com Figure 1: ECHOS analysis of 2D-NMR spectra for quantification of similarity between originator and biosimilar. Box indicate how specific peak differences can be identified from the ECHOS analysis.