Chapter IV Effects of operational parameters during UF on the
3.3 Conformational stability
ATR FT-IR temperature ramps are known to be capable to evaluate the thermal stability of proteins. The protein melting temperature (Tm) which is defined as the temperature
where equal amounts of native and denatured structure exist in equilibrium can be deter- mined by monitoring the position and intensity of the amide I component frequencies as a function of temperature. In this study Tm was used to investigate if changes in conforma-
tional stability occur depending on the operational conditions used in UF, thus influencing aggregation during storage.
MAbs show two characteristic bands in the second derivative spectrum at around 1639 and 1690 cm-1 reflecting the presence of intramolecular ß-sheet (Chang et al. 2005; Cleland et al. 2001). Conformational changes are indicated by a reduced band at around 1639 cm-1
and a concomitantly arising strong temperature-induced band at 1615-1625 cm-1
. Moreover, a weak intensity peak at around 1690 cm-1
arises. All these spectral changes are assignable to the formation of intermolecular β-sheets which was fund to be mainly pre- sent in aggregated protein molecules (Pelton and McLean 2000). Figure IV-13A-C shows the second derivative spectra of the heated solutions at 90 mg/ml derived from the differ- ent UF methods. In the case of the used IgG, the band at 1637 cm-1
was found to strongly decrease and the band at 1624 cm-1
was found to strongly increase with higher tempera- ture.
3 Results and discussion
Figure IV-13: ATR FT-IR second derivative spectra of mAb solutions concentrated to 90 mg/ml by using different UF methods
Arrows mark the direction of spectral changes of the bands at 1637 cm-1 and
1624 cm-1 with increasing temperature from 25 (bolded black line) to 90 °C
(bolded grey line); ∆p 1.2 bar (A), ∆p 3.0 bar (B) and optMeth (C).
Different methods can be in general applied to determine Tm of a protein from the FT-IR
spectra. Based on direct plots of the relative intensity values of the decreasing native band and the increasing temperature induced band from the second derivative spectra, the mid- point of the thermal transition can be identified. For this, the inflection point of both curves can be determined which is described by Dong et al. (1997) for Factor XIII and by Kendrick et al. (1998b) for huIFN-γ. In addition, the intersection between the tempera- ture-induced band and the disappearing frequency of the native state can be determined, as reported by Matheus et al. (2006a) for different proteins including an IgG1.
Figure IV-14 shows the changes in intensity in the second derivative spectra for the frequencies at 1637 cm-1
and 1624 cm-1
with increasing temperature up to 90 °C for the three concentrates generated by using different UF protocols. With increasing temperature a continuous increase in relative intensity of the native amide I band was observed which was accompanied by a continuous decrease in relative intensity at 1624 cm-1
. Both curves show a sigmoid shape with a clearly defined inflection point. The inflection point of the curves was calculated by using the Origin software (Origin 7.5 SR5, OriginLab Corpora- tion, Northampton, USA) applying a sigmoid fit according to the Bolzmann equation (Markovic et al. 2007). Table IV-2 summarizes the determined melting temperatures based on the three different interpretation approaches described above.
1580 1600 1620 1640 1660 1680 1700 wave number [cm-1] a rb it ra ry u n it s B A C 1637cm-1 1624 cm-1 1580 1600 1620 1640 1660 1680 1700 wave number [cm-1 ] 1580 1600 1620 1640 1660 1680 1700 wave number [cm-1] 1689cm-1 1580 1600 1620 1640 1660 1680 1700 wave number [cm-1] a rb it ra ry u n it s B A C 1637cm-1 1624 cm-1 1580 1600 1620 1640 1660 1680 1700 wave number [cm-1 ] 1580 1600 1620 1640 1660 1680 1700 wave number [cm-1] 1689cm-1
Figure IV-14: Changes in intensity in the ATR FT-IR second derivative spectra at 1637 cm-1 (black squares) and 1624 cm-1 (grey squares) during heating up to
90 °C of a 90 mg/ml mAb solution concentrated by using different UF methods
∆p 1.2 bar (A), ∆p 3.0 bar (B) and optMeth (C); the respective sigmoid fit accord- ing to the Bolzmann equation is indicated by the solid line.
A Tm of around 70 °C was observed which correspond to the values for IgGs that have been
reported in literature (Matheus et al. 2006a; Matheus et al. 2006b; Vermeer et al. 1998; Ver- meer and Norde 2000). Considering the different interpretation approaches to determine Tm,
quite similar values were obtained. In general, the values for Tm identified by using the inflec-
tion point of the decreasing band are always lower than the values based on the intensities of the increasing frequency. For both approaches, as well as for the interpretation by using the intersection between the denatured and native frequency, consistently higher values for Tm
were identified for the concentrates generated by using the optimized UF method. B A C 50 55 60 65 70 75 80 85 90 95 re la ti v e i n te n s it y temperature [°C] 50 55 60 65 70 75 80 85 90 95 re la ti v e i n te n s it y temperature [°C] 50 55 60 65 70 75 80 85 90 95 re la ti v e i n te n s it y temperature [°C] B A C 50 55 60 65 70 75 80 85 90 95 re la ti v e i n te n s it y temperature [°C] 50 55 60 65 70 75 80 85 90 95 re la ti v e i n te n s it y temperature [°C] 50 55 60 65 70 75 80 85 90 95 re la ti v e i n te n s it y temperature [°C]
3 Results and discussion
Tm (FT-IR) [°C]
Inflection point using sigmoid fit (Boltzmann equation) Method Protein concentration Increasing band at 1637 cm-1 Decreasing band at 1624 cm-1 Intersection between in- and decreasing band
before UF 5 mg/ml 69.2 ± 0.2 69.6 ± 0.2 69.9
∆p 1.2 bar 90 mg/ml 70.9 ± 0.2 69.8 ± 0.2 71.2
∆p 3.0 bar 90 mg/ml 70.2 ± 0.1 69.4 ± 0.2 70.1
optMeth 90 mg/ml 71.5 ± 0.2 70.5 ± 0.2 71.9
Table IV-2: Melting temperature (Tm) for mAb solutions at 90 mg/ml determined from
ATR FT-IR second derivative spectra by using three different interpreta- tion approaches
Results of sigmoid fitting are listed with ± 1 standard error of the non-linear re- gression.
Concomitantly, a consistently higher Tm was recognized for the concentrated solutions at
90 mg/ml compared to the Tm values of the solutions at 5 mg/ml. This suggests that con-
formational stability regarding secondary structure is affected by the UF method used, but in general, a higher total protein concentration leads to an increase of the melting tempera- ture as well. This is in accordance with current literature reporting that concentrated protein solutions are protected against conformational changes. The rate of denaturation of several proteins exposed to heat and ethanol has been found to be reduced in the presence of sig- nificant concentrations of up to 100 mg/ml of other heat stable proteins (Minton 2000; Minton et al. 1982). The addition of dextran at high concentrations stabilizes lysozyme and cytochrome C with respect to thermal denaturation (Sasahara et al. 2003). Matheus and co- workers (2006a) reported that an IgG1 formulation at 100 mg/ml showed nearly no differ- ence in Tm when the pH was changed from 5 up to 8 although aggregation was clearly fa-
vored at pH 8.
An explanation is provided by the excluded volume theory arguing that in the pres- ence of highly concentrated macromolecules the unfolding of proteins should be reduced due to facing a crowded or so-called volume-occupied system (Alford et al. 2008a; Hall and Minton 2003; Harn et al. 2007). Due to steric- and charge-based repulsion of the mac- romolecules, the protein molecules exclude volume to another thus resulting in an increase in free energy. As volume occupancy increases, compact protein conformations of the native state become increasingly energetically favored over extended conformations of
equal volume, present in the denatured state (Minton 1980). Therefore, the native confor- mation of a protein becomes more stable relative to any less compact denatured state.
It is concluded that the formation of aggregates in highly concentrated solutions is rather controlled by a homogeneous nucleation and growth process than by partially un- folded molecules, since the protein was shown to be in general protected against conforma- tional changes at a higher total protein concentration. This is in accordance with the litera- ture, since nucleation and growth phenomena were mainly recognized when the native con- formation of the protein was preserved (see chapter I). The increase in Tm at a high protein
concentration of 90 mg/ml did not result in a decreased formation of aggregates during storage below the melting temperature. Quite the contrary was recognized, since aggregation was enhanced with increasing total protein concentration in the solution. The applied UF method seems to significantly affect the conformational stability of the protein, but these results did not correspond to the aggregation behavior during performed stability studies. No significant differences regarding the level of several types of aggregates in the differently processed concentrates were observed during storage below Tm.
4
Summary and conclusion
In this chapter, the physical stability of highly concentrated IgG bulks derived from differ- ent UF methods was investigated. The three stage protocol using the optimized opera- tional conditions presented in chapter III showed the lowest level of larger aggregates be- fore and during storage at elevated temperature, at 2-8 °C and after freezing and thawing.
After 0.2 µm-filtration of the concentrates no differences regarding the level of larger subvisible protein aggregates detected by turbidity measurements and LO were observed depending on the UF method used. This was recognized during storage at elevated tem- perature, at 2-8 °C as well as after freezing and thawing in three cycles. Almost all pro- teinaceous particles were removed by filtration and did not reappear during storage. There- fore, the improved quality of the bulk derived from the optimized UF method regarding the level of proteinaceous particles is no longer relevant after the filtration of the concen- trated bulk.
As regards soluble HMWs, no significant differences between the several unfiltered concentrates were observed neither before, nor after storage. Only after 6 weeks at 40 °C
4 Summary and conclusion
the filtered concentrates revealed a significantly higher level of HMWs generated by using the ∆ 3.0 bar method. In general, during freezing and thawing in three cycles a significantly increased level of HMWs was detected. Both, the filtered and unfiltered solutions at 90 mg/ml showed an increase in HMWs up to three times compared to the solutions at 5 mg/ml. Since the level of HMWs was quite stable at 1 % when the concentrates were stored at 2-8 °C, this is the recommended storage condition for the unformulated highly concentrated bulk material.
The filtered solutions stored at elevated temperature showed a higher level of soluble aggregates compared to the concentrates which remained unfiltered during storage. This was attributed to the already existing larger subvisible aggregates in the unfiltered solutions preventing the formation of the smaller soluble aggregate species in accordance with the theory of homogeneous nucleation and growth.
The secondary structure was found to be affected by the UF methods used. The con- centrates generated by using the optimized method showed a consistently higher melting temperature which was evaluated by using three different interpretation approaches based on temperature dependent ATR FT-IR second derivative spectra analysis. Moreover, the highly concentrated protein solutions showed consistently higher Tm values compared to
the solutions with a protein concentration of only 5 mg/ml. In contrast, aggregation dur- ing storage was in general enhanced at a higher total protein concentration, independent from the UF method used. Thus, aggregation during storage at higher total protein con- centrations seems to be rather based on homogeneous nucleation and growth than on a perturbation in secondary structure. The preservation of the native conformation of the protein does not seem to be efficient enough to ensure the physical stability of the concen- trated bulk. Physical stability encompasses in fact conformational and colloidal stability and the latter seems to be the more decisive factor regarding the storage stability of the highly concentrated solutions.