Category: Formulation and Quality
Purpose: Biologics products including monoclonal antibodies, therapeutic proteins, and vaccines. The global biologics market is anticipated to be worthy of $399.5 billion by the year 2025, and monoclonal antibodies accounted for 43% of the market. The gastrointestinal-targeted oral delivery of antibody via tablets have some advantages over the parental route of administration consider its better patient compliance, easy administration, and high manufacturing efficiency.
We aim to study the effects of tableting compression force, particle size and relative humidity on the IgG stability. Mechanical stresses such as the compression force during the tableting process and relative humidity during the storage could induce protein conformational change and aggregation, which may cause immunogenicity and affect antibody functionality. Analytical and biophysical techniques such as UPLC-SEC, DLS, CD, FTIR and nano-DSC have been used in this study for the characterization of IgG tablet stability.
Methods: To test the size and compaction effects, lyophilized IgG powders were separated into two particle size ranges: 350-500 μm & >500μm, and compressed under various pressures (50-350 MPa) using a material testing machine. IgG powders were keep in different relative humidity (32%, 52%, 75%, 93% RH) for 1 month and compressed into tablets at two pressures (100 MPa and 200 MPa) for study the RH and pressure effect. Soluble aggregates from the reconstituted protein samples were characterized via UPLC-SEC and DLS. Reconstituted protein samples were analyzed via FTIR and secondary structures were predicted via studying amide I band (1600 to 1700 cm-1) using PLS Toolbox and MATLAB. CD was also applied to study the protein secondary structure changes. The thermal stability of reconstituted proteins were analyzed by Nano-DSC.
Results: For the characterization of soluble aggregates, our preliminary results from both UPLC-SEC and DLS showed that different compression forces did not induce significant aggregation during the tableting process with monomer percentage kept around 80-90%; however, tablets made with smaller particle size (350-500 μm) showed slightly lower monomer percentage compared with those made with larger particle size ( >500 μm). Interestingly, UPLC-SEC analysis showed higher percentage of soluble aggregates in reconstituted protein samples with up to ~37% aggregates (RH 93%) in higher storage relative humidity compared with ~27% from control group. DLS analysis showed similar trend with higher percentage of aggregates up to 52% in higher storage relative humidity (RH 93%) compared with 5% in control group (Fig 1). CD analysis of secondary structure showed significantly reduced helix structure (from 25.1% in control IgG powders to 1.1% in tablets made with higher compression force), and slightly increased β strand and β turn structures, as well as significantly increased unordered structures (from 0% in control IgG powder to ~40% in tablets made with higher compression force). However, CD analysis did not reveal any significant secondary structure changes for different relative humidity tablets. Solid state FTIR analysis of Amide I region with principal component analysis (PCA) of tablets made with IgG powders in different storage relative humidity chambers and with 200 mPa compression force was performed. Using PCA, the score plot showed clustering of RH 32% & Control and RH 75% & 93%, which indicates they share similar characteristics. Loading plot of PC1 (responsible for 74.62% of variation) indicated the peaks around 1609, 1622, 1635, 1652, 1666 and 1682 cm-1 were positively correlated with the sample groups and peaks around 1629, 1644, 1660, 1675 and 1690 cm-1 were negatively correlated with the sample groups. FTIR peaks around 1609, 1622 and 1682 are correlated with protein aggregation. FTIR peaks around 1635, 1652 and 1666 cm-1 are correlated with parallel β-sheet, α-helix, and β-turn respectively. FTIR peaks around 1629/1690, 1644, and 1666/1675 cm-1 are correlated with anti-parallel β-sheet, unordered, β-turn, respectively (Fig 2). Nano-DSC analysis showed three different melting temperatures with Tm1 around 71 oC, Tm2 around 77 oC and Tm3 around 83 oC. Tm1, Tm2, Tm3 are assigned to correspond to Fc-CH2, Fab, Fc-CH3 regions, respectively; due to their inherent thermal stability. Nano-DSC analysis did not observe significant Tm changes corresponding to different compression forces and storage relative humidity (Fig 3).
Conclusion: Based on the UPLC-SEC and DLS data, the commonly used compression pressures applied to make the IgG tablets did not induce significant aggregation; however, higher storage relative humidity induced significant aggregation. Based on the CD data, an interchange of helix structure to β structures and unordered structures may due to the β sheets and unordered structure provide better flexibility that may be able to protect the IgG during the tableting compaction, which reduces intermolecular distance. Solid-state FTIR data showed increased aggregation bands corresponding with higher relative humidity. Nano-DSC analysis showed the thermal stability was not affected by either compression force or relative humidity.
Yuwei Lu– University of Maryland, Baltimore, Maryland
Changquan Calvin Sun– Minneapolis, Minnesota
Chenguang Wang– Postdoc, University of Minnesota, Minneapolis, Minnesota
Stephen Hoag– Professor, University of Maryland School of Pharmacy, Baltimore, Maryland
Yuwei Lu– University of Maryland, Baltimore, Maryland