Chapter II Stabilization of highly concentrated antibody-
3. Results and Discussion
3.2 Influence of hydroxypropyl-beta-cyclodextrin on the stability of dilute and highly concentrated
3.2.5 Thermal analysis
The conformational stability of both IgGs in the presence of different excipients was analyzed by determining the respective protein melting behavior. Firstly, melting points of 100 mg/mL IgG A-formulations were measured by FTIR-analysis, which is known as material saving, non-invasive method, and applicable for direct measurement at high protein concentration. [56, 57] Melting point analysis by FTIR was considered most suitable for proteins with initially high intramolecular β-sheet structures. [56] Second derivative spectra of 100 mg/mL IgG A in the native (25°C) as well as thermally denatured (95°C) conformation are illustrated in Figure II.18. The main band at 1636 cm-1 in the native form indicates the predominant presence of intramolecular β-sheets. With increasing temperature, the main band at 1636 cm-1
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disappears, and a new band at 1622 cm-1 is formed which refers to the origin of intermolecular β-sheet structures. [56] For determination of the melting curves, respective adsorption ratios between 1636 cm-1 and 1622 cm-1 are calculated for each measured temperature point, and were plotted against the corresponding temperature (Figure II.19).Figure II.18: Changes in second derivative FTIR-spectra of 100 mg/mL IgG A-solution without
excipient before (solid line) and after (dotted line) heating from 25°C to 95°C.
The melting curves for each of the tested 100 mg/mL IgG A-formulations (without excipient, with addition of 25 mM HPβCD, and with addition of 0.1% polysorbate 80) show a perfect overlay (Figure II.19) which is also mirrored in the corresponding melting temperatures in Table II-1. Therefore, neither the addition of 25 mM HPβCD, nor the presence of 0.1% polysobate affects the conformational stability of IgG A in the highly concentrated solution.
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Figure II.19: Melting curves of 100 mg/mL IgG A-formulations containing no excipient, HPβCD 25 mM,
and polysorbate 0.1% as determined by FTIR-measurements.
sample melting temperature Tm
without excipient 74.45°C ± 0.20 HPβCD 25 mM 74.32°C ± 0.08 polysorbate 0.1% 74.44°C ± 0.56
Table II-1: Melting points of 100 mg/mL IgG A-formulations as determined from the inflection point
of the melting curves in Figure II.19.
In general, measuring the protein melting behavior by FTIR-temperature ramps provides the great benefit of low sample volumes, and the capability to measure in a broad concentration range. [56] However, the applicability is limited by a comparably long duration of analysis and therefore, further measurements with highly concentrated IgG-formulations were performed by using a common DSC-device. Additionally, low concentrated protein samples were investigated by Microcal VP-DSC.
The thermograms of 1.8 mg/mL and 100 mg/mL samples (Figure II.20) show two melting peaks, which are related to the Fab and Fc parts of the antibody. [58] Interestingly, IgG A
exhibited a predominant first (Tm1) and a much smaller second (Tm2
)
melting point whereasIgG B shows a smaller Tm1 and a dominating Tm2. Thermograms of the respective samples
containing 100 mM HPβCD are integrated into the graphs to illustrate the influence of the cyclodextrin on the melting behavior (Figure II.20). In all tested formulations, the cyclodextrin- formulation resulted in a shift of the melting-temperature to lower values.
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(A) IgG A – 1.8 mg/mL (B) IgG A – 100 mg/mL
(C) IgG B – 1.8 mg/mL (D) IgG B – 100 mg/mL
Figure II.20: Thermograms of (Left) 1.8 mg/mL IgG A (Figure A) and IgG B (Figure C) and (Right)
100 mg/mL IgG A (Figure B) and IgG B (Figure D) in the formulation without excipient (black) and with addition of 100 mM HPβCD (grey) as determined by DSC.
The main melting-temperatures (that of the predominant peak) of all tested formulations are illustrated in Figure II.21. The results show that sucrose increased the Tm - except in the
100 mg/mL IgG A formulation (Figure II.21B) - which is in accordance with the preferential exclusion phenomenon. [57] By contrast, polysorbate had no or only a minor influence on Tm.
Meanwhile, the low concentrations of HPβCD did not or only marginally affect the melting temperature, but a continuous decrease in Tm was observed with increasing cyclodextrin
concentrations. Here, the decrease in melting temperature was more obvious for IgG A than for IgG B with 1,5°C- (Figure II.21A) and 3°C-decrease (Figure II.21B) in the 100 mM formulation for IgG A, and roughly 0.4°C-decrease in the 100 mM formulations for IgG B (Figure II.21C and D). Since the decrease in melting temperature by addition of HPβCD might be ascribed to an interaction between the cyclodextrin and the IgG, different melting
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behavior of IgG A and IgG B after addition of cyclodextrin indicates a possible difference in the interaction between the two IgGs and HPβCD. Comparing the Tm of dilute solutionswithout excipients vs. concentrated protein solution, no significant difference was seen for IgG B whereas for IgG A, Tm is lower for the dilute sample compared to the concentrated
one. A similar observation was reported for thermal analysis of two monoclonal antibodies, which showed a 2-3°C higher melting point when increasing the protein concentration from 1 to 100 mg/mL. [18] Stabilization of proteins due to higher concentrations was ascribed to the excluded volume theory in crowded environments which hinders the protein from unfolding. [18, 59]
The results of the melting temperature (Figure II.21) are reflected in the outcome of quiescent storage at 50°C (Figure II.17), where the presence of increasing HPβCD- concentrations lead to a gradually destabilization of the protein. Predictability of protein stability during long time storage based on Tm-measurements was already shown before for a monoclonal antibody. [60] Interestingly, despite the gradual decrease in conformational protein stability in the presence of increasing HPβCD-concentrations, the resistance against mechanically induced aggregation (see previous shaking and stirring experiments) was increased.
The mechanism of protein stabilization against mechanical agitation observed with cyclodextrins is highly debated in the literature. Many investigators report the binding of cyclodextrins to hydrophobic residues as the underlying cause of stabilization. [61, 62] For instance, cyclodextrins can form host-guest complexes with the hydrophobic amino acids (mainly Phe, Tyr and Trp) of some proteins, such as hGH and LHRH [21, 63-66], leading to inhibition of aggregation. Others hold the surface activity of some cyclodextrins (e.g. HPβCD) responsible for their stabilizing activity. For instance, the effectiveness of HPβCD in reducing interfacially-induced precipitation of porcine growth hormone was ascribed to the surface activity of HPβCD. [67] In another study, the proposed relationship between the interfacial stabilization of rh-GH by HPβCD and surface activity of HPβCD was substantiated by correlating increasing degrees of substitution of HPβCD (that translate into increasing surface activity) to reduced amounts of aggregates in vortexed rh-GH formulations. [47] In the current study, the progressive increase in stability with increasing concentrations of HPβCD concentrations point out to the possibility of a direct interaction between IgG and the cyclodextrin rather than a surface effect.
A cyclodextrin-induced decrease in melting temperature was reported for globular proteins in the presence of α-cyclodextrin, whereas the shift in melting temperature was ascribed to a weak non-covalent interaction between the cyclodextrin and hydrophobic side chains of the unfolded protein. [68] In a further study with lysozyme, presence of hydrophilic cyclodextrins in a concentration of 100 mM lead to a significant reduction of the protein melting
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temperature via stabilizing an unfolded protein conformation, but chemically induced protein- aggregation was significantly reduced. [69] The hypothesis of a binding between HPβCD and native/unfolded IgG A and IgG B was studied in detail by quartz crystal microbalance and light-scattering experiments, and will be extensively discussed in chapter IV.(A) IgG A – 1.8 mg/mL (B) IgG A – 100 mg/mL
(C) IgG B – 1.8 mg/mL (D) IgG B – 100 mg/mL
Figure II.21: Protein melting temperature of (Left) 1.8 mg/mL IgG A (Figure A) and IgG B (Figure C)