protein species accompanying the monomeric antibody, such as fragments and aggregates.
Repeated samplings were performed for each chosen stress condition and hence the progress in size distribution of soluble species was monitored. Figure 4-2 provides an overview of the total recoveries after all stress conditions determined by SEC.
Figure 4-2 – Recoveries of soluble protein species after stressing muAb – determined by SEC.
The total recoveries after stirring (red), shaking (black), light exposure (blue) and heating (green) are shown compared to the incubation time (time axis not true to scale). A triplicate of samples was analyzed and the error bars represent the standard deviation of n=3.
Stirring the murine antibody with 400 rpm initiated a considerable loss of total protein recovery in SEC. After 24 hours of stirring 43 ± 19 % of the original protein content was detected yet. Reviewing the tremendous increase in subvisible particles and turbidity in these samples, the formation of insoluble protein species is suggested. The centrifugation of the samples prior to injection to the SEC column separated these species from the solution finally injected.
The aggregation studies with the human IgG1 antibody previously described also revealed a formation of insoluble protein species from shaking, though not as incisive as from stirring. Shaking the murine antibody with 400 rpm also initiated a steady loss of total protein recovery in SEC. After 48 hours of shaking 84 ± 4 % of the injected protein mass was detected
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by UV absorption at 280 nm. Similarly to stirring, the formation of subvisible particles discovered by LO and turbidity measurements confirm the findings. Both methods, shaking and stirring, did not initiate the formation of fragments or soluble aggregates feasible to be separated by SEC. The UV 280 nm chromatograms did not show any new emerging peaks (data not shown).
During storage of the antibody at an elevated temperature of 50°C the total protein recovery decreased within the first week to 77 ± 4 % (see Figure 4-2). Subsequently, it approximately remains on this level during the next two weeks, and further drops until day 27 reaching 59 ± 1 % of total recovery.
The total recoveries determined in the light exposed samples revealed a constantly decreasing profile up to the final incubation time of 72 h.
Comparing the kinetics in the loss of total recovery between all stress conditions obviously stirring appears to be the fastest method to generate huge amounts of insoluble species which cannot be detected by SEC anymore. A drastic but slower loss of total recovery was determined after light exposure as well, whereas shaking and storage at 50°C revealed a very slow reduction of total recovery within the first days.
Figure 4-3 – Distribution of soluble protein species in muAb samples stored at 50°C determined by UV detection after SEC separation.
The UV chromatograms of the native muAb (red line) and the muAb after 27d of storage at 50°C (black line) are scaled on the left y-axis. The molar masses determined are scaled on the right axis.
The incubation at 50°C additionally led to the formation of additional species of lower and higher molecular weight detected by UV 280 nm. The molar mass determination of this peak indicated it to be formed by fragments (~42 kDa). The main peak comprised 85 % of the in total recovered soluble protein and represented the monomeric muAb approved by a molar mass determination of ~138 kDa. Furthermore, two species of higher molecular weight could be
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differentiated due to retention time (see Figure 4-3). A small fraction eluting around 27 – 28 minutes was detected in UV and supposed to be dimers and/or trimers, though for molar mass determination by MALLS the peak was undersized. Besides, oligomers were detected, eluting in a broad peak prior to the dimers/trimers and the monomer. A molar mass of ~2 MDa was determined in a narrow section in the center of the peak. A broad size distribution of species is suggested to form this peak. Figure 4-4 provides the overview of the different species formed during storage at 50°C. The maximum of soluble aggregates was detected after 2 weeks of storage, subsequently dropping simultaneous to the loss of total recovery.
Figure 4-4 – Distribution of soluble protein species after heating muAb – determined by SEC.
The total recovery of soluble species is shown by the red squares, scaled on the right y-axis. The white bars represent the amount of species of lower molecular weight (fragments), the striped bars represent the monomer, the checkered bars represent small aggregates and the black bars represent oligomers.
The error bars show the standard deviations of n=3 samples.
Soluble aggregates of the murine IgG2c antibody were as well generated by light exposure. The samples irradiated by light feature the quick formation of soluble species of both lower and higher molecular weight. The histidine containing formulation buffer implied the rising of a variety of peaks eluting between 38 and 52 minutes that are also seen in control samples of light exposed buffer. Figure 4-5 exemplarily displays the chromatogram after 72 h of light exposure overlaid to the native muAb sample and the shielded control sample. The formulation buffer degraded by light exposure is shown as well.
The majority of the total area under the curve after 72 h of light exposure eluted prior to the monomer peak. These larger molecular weight species are separated in a sharp peak eluting after 15 minutes and a broad peak eluting between 17 and 28 minutes. The molar mass within these peaks revealed approximations of 1.5 to 3 MDa (see Table 4-3). Besides the generation of huge amounts of aggregates, the originally detected peak representing fragments of muAb raised as well. Within the control samples (shielded from light but exposed to the
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elevated temperature of ~35°C) 2.5 % lower molecular weight species but no species of higher molecular weight (aggregates) were detected.
Figure 4-5 – Distribution of soluble protein species of muAb samples after light exposure determined by UV detection after SEC separation.
The UV 280 nm chromatograms represent the native murine antibody (black line), the control samples shielded from light but incubated 72h (blue line), and the sample after 72h of light exposure (red line). The grey line shows the chromatogram of light exposed formulation buffer (not true to scale).
Table 4-3 – SEC: Molecular weight approximations and area percentages (UV) of muAb samples.
Sample LMW (Fragments) Monomer HMW 1 HMW 2
MW % MW % MW % MW %
Native muAb 50 kDa 1.2 % 124 kDa 98.8 % - - - -
Control shielded
from light 38 kDa 2.5 % 123 kDa 97.5 % - - - -
72 h light
exposure n/a 4.0 % n/a 9.2 % 2.7 MDa 50.4 % 1.6 MDa 36.4 %
The distribution of soluble protein species as a function of incubation time is shown in Figure 4-6. Throughout the investigated 72h of light exposure the majority of remaining soluble protein species is represented by higher molecular weight species (aggregates) which were not detectable before irradiation. After the entire time of light exposure, 88.5 ± 0.2 % of aggregates were detected within the 24.3 ± 0.7 % of total protein that could be recovered. The rest was already precipitated into particles. The percentage of aggregates steadily increased during irradiation, whereas the monomer content coincidentally decreased. At the same time the amount of fragments in the samples remains more or less constant between 5.6 and 8.8 %.
HMW 1 HMW 2 LMW Buffer degradation products Monomer
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Figure 4-6 – Time dependent distribution of soluble muAb species determined by SEC after light exposure.
The left y-axis scales the recovery of the different species: fragments (white bars), monomer (striped bars) and aggregates (black bars). The right y-axis scales the total recovery displayed by the red triangles. The error bars represent the standard deviation of triplicate measurements.
4.3.3 Modifications in the structure of the murine IgG2c antibody (muAb)