European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 299–309
Contents lists available at ScienceDirect
European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb
Research paper
Agitation-induced aggregation and subvisible particulate formation in model proteins Murali Jayaraman, Patrick M. Buck, Arun Alphonse Ignatius, Kevin R. King, Wei Wang ⇑ Pharmaceutical Research and Development, Pfizer Inc, Chesterfield, MO, USA
a r t i c l e
i n f o
Article history: Received 1 September 2013 Accepted in revised form 17 January 2014 Available online 23 January 2014 Keywords: Protein Agitation Particle Aggregation Stability Unfolding
a b s t r a c t The kinetics of agitation-induced subvisible particle formation was investigated for a few model proteins – human serum albumin (HSA), hen egg white lysozyme (HEWL), and a monoclonal antibody (IgG2). Experiments were carried out for the first time under relatively low protein concentration and low agitation speed to investigate the details of subvisible particle formation at the initial phase of aggregation ( HSA > HEWL, while HSA has the largest hydrophobic patch, followed by IgG2 and HEWL. The surface hydrophobic properties of these model proteins upon agitation were measured by SYPRO orange fluorescence. Fig. 6 shows the relative fluorescence intensity of protein samples with time during agitation. At time zero, significant fluorescence (85 ± 8 a.u.) was
The thermal stability of the three model proteins before and after agitation was compared by both DSC and DSF (Fig. 7). The DSC curve of HSA before agitation showed three unfolding transitions with Tm1 at 56.6 °C, Tm2 at 70.1 °C and Tm3 at 80.4 °C respectively. The corresponding transitions of the 7-h agitated sample were observed at 56.7 °C, 70.0 °C and 82.3 °C, respectively. The agitation stress did not alter the Tm and enthalpy (DH) values of the first two transitions, however, the Tm3 was slightly increased with a significantly lower DH. The DSF curve of the same HSA sample exhibited three unfolding transitions (Th – hydrophobic melting temperature) at 58.7 °C, 68.9 °C and 77.0 °C, respectively. The 7-h agitated sample exhibited similar values but the fluorescence intensity was significantly increased (22%). Control experiments carried out with free dye exhibited very minimal fluorescence change at the melting temperature in the DSF measurements. The DSC thermogram of HEWL showed a single melting transition at 76.6 °C. There was no difference in Tm and DH values between the unagitated and 7-h agitated HEWL samples. Similarly, a single unfolding transition was observed at 72.3 °C by DSF measurement, which is a few degrees lower than that determined by DSC. In addition, the relative fluorescence intensities at the onset unfolding temperature were about 7% higher for the agitated HEWL sample compared to the unagitated sample. The DSC thermogram of IgG2 molecule exhibited three transitions at 72.9 °C, 82.1 °C and 87.1 °C, respectively. Agitation for 7 h showed a negligible difference in enthalpy and melting temperatures. DSF data of IgG2 showed similar number of transitions but the Th values are 2–6 °C lower than the DSC data. Similar to HEWL, the agitated IgG2 sample exhibited a higher fluorescence intensity at the onset unfolding temperature. The significant increase in fluorescence intensity for HSA (Table 1) suggests an increase in exposed hydrophobic surfaces as a result of agitation stress. Among the three model proteins, HSA had the lowest Tm value (56.6 °C) (Table 2), suggesting that it is the least thermally stable protein, at least for one domain. The Tm1 value of HSA obtained by DSC is 2.2 °C lower than the hydrophobic melting temperature by DSF (Fig. 8). In comparison, the highest Tm of HSA and all the Tm’s of the other two model proteins are a few degree higher than those by DSF. These differences in the thermal unfolding pattern are possibly due to the variations in hydrophobic exposure and unfolding of different domains. 4. Discussion Physical instability of different proteins may result in formation of soluble and insoluble aggregates in liquid protein formulations. However, the exact mechanism of agitation-induced aggregation at the air–water interface remains unclear. To better understand this process, we have employed multiple techniques on three model proteins, HSA, HEWL and IgG2. These proteins have diverse
304
M. Jayaraman et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 299–309
Fig. 5. Reversibility of HSA (A and D), HEWL (B and E) and IgG2 (C and F) particles formed during agitation on 5 fold dilution either in 200 mM sodium phosphate buffer (pH 7.0) containing 50 mM NaCl (A–C) or in 10 mM sodium acetate formulation buffer (pH 5.5) (D–F).
structural and physical properties (Molecular weight, pI, surface hydrophobicity and thermal stability). Moreover, these model proteins have been previously investigated for their aggregation behavior under agitation and other stress conditions [24–26].
4.1. Mass balance during agitation All the model proteins lost a significant amount of monomer with formation of soluble aggregates (HSA and HEWL only) and
305
M. Jayaraman et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 299–309 Table 1 Average fluorescence intensities from DSF melting curves of the unagitated and 7-h agitated samples of HSA, HEWL and IgG2. Protein
Agitation time (h)
Average (three samples) ± SD
HSA
0 7
4470 ± 112 5493 ± 260
HEWL
0 7
2023 ± 4 2018 ± 3
IgG2
0 7
2071 ± 9 2069 ± 14
subvisible particles upon agitation. When the percentage of remaining protein monomers is compared with the sum of soluble aggregates by SEC-HPLC and insoluble proteins converted into subvisible particles detected by HIAC, it is clear that the total number does not add up to 100%. The higher percentage of monomer loss than what can be accounted for by SEC and HIAC could be attributed to several possibilities. First, particles of submicron range (128 nm aggregates immediately after agitation. One, however, has to
understand that these size estimations as measured by DLS may not be very accurate, as the DLS method cannot distinguish well protein monomers from dimers and trimers in solution and is semi-quantitative in estimating higher molecular weight aggregates [32,33]. Nevertheless, the lack of smaller oligomeric aggregates (
Contents lists available at ScienceDirect
European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb
Research paper
Agitation-induced aggregation and subvisible particulate formation in model proteins Murali Jayaraman, Patrick M. Buck, Arun Alphonse Ignatius, Kevin R. King, Wei Wang ⇑ Pharmaceutical Research and Development, Pfizer Inc, Chesterfield, MO, USA
a r t i c l e
i n f o
Article history: Received 1 September 2013 Accepted in revised form 17 January 2014 Available online 23 January 2014 Keywords: Protein Agitation Particle Aggregation Stability Unfolding
a b s t r a c t The kinetics of agitation-induced subvisible particle formation was investigated for a few model proteins – human serum albumin (HSA), hen egg white lysozyme (HEWL), and a monoclonal antibody (IgG2). Experiments were carried out for the first time under relatively low protein concentration and low agitation speed to investigate the details of subvisible particle formation at the initial phase of aggregation ( HSA > HEWL, while HSA has the largest hydrophobic patch, followed by IgG2 and HEWL. The surface hydrophobic properties of these model proteins upon agitation were measured by SYPRO orange fluorescence. Fig. 6 shows the relative fluorescence intensity of protein samples with time during agitation. At time zero, significant fluorescence (85 ± 8 a.u.) was
The thermal stability of the three model proteins before and after agitation was compared by both DSC and DSF (Fig. 7). The DSC curve of HSA before agitation showed three unfolding transitions with Tm1 at 56.6 °C, Tm2 at 70.1 °C and Tm3 at 80.4 °C respectively. The corresponding transitions of the 7-h agitated sample were observed at 56.7 °C, 70.0 °C and 82.3 °C, respectively. The agitation stress did not alter the Tm and enthalpy (DH) values of the first two transitions, however, the Tm3 was slightly increased with a significantly lower DH. The DSF curve of the same HSA sample exhibited three unfolding transitions (Th – hydrophobic melting temperature) at 58.7 °C, 68.9 °C and 77.0 °C, respectively. The 7-h agitated sample exhibited similar values but the fluorescence intensity was significantly increased (22%). Control experiments carried out with free dye exhibited very minimal fluorescence change at the melting temperature in the DSF measurements. The DSC thermogram of HEWL showed a single melting transition at 76.6 °C. There was no difference in Tm and DH values between the unagitated and 7-h agitated HEWL samples. Similarly, a single unfolding transition was observed at 72.3 °C by DSF measurement, which is a few degrees lower than that determined by DSC. In addition, the relative fluorescence intensities at the onset unfolding temperature were about 7% higher for the agitated HEWL sample compared to the unagitated sample. The DSC thermogram of IgG2 molecule exhibited three transitions at 72.9 °C, 82.1 °C and 87.1 °C, respectively. Agitation for 7 h showed a negligible difference in enthalpy and melting temperatures. DSF data of IgG2 showed similar number of transitions but the Th values are 2–6 °C lower than the DSC data. Similar to HEWL, the agitated IgG2 sample exhibited a higher fluorescence intensity at the onset unfolding temperature. The significant increase in fluorescence intensity for HSA (Table 1) suggests an increase in exposed hydrophobic surfaces as a result of agitation stress. Among the three model proteins, HSA had the lowest Tm value (56.6 °C) (Table 2), suggesting that it is the least thermally stable protein, at least for one domain. The Tm1 value of HSA obtained by DSC is 2.2 °C lower than the hydrophobic melting temperature by DSF (Fig. 8). In comparison, the highest Tm of HSA and all the Tm’s of the other two model proteins are a few degree higher than those by DSF. These differences in the thermal unfolding pattern are possibly due to the variations in hydrophobic exposure and unfolding of different domains. 4. Discussion Physical instability of different proteins may result in formation of soluble and insoluble aggregates in liquid protein formulations. However, the exact mechanism of agitation-induced aggregation at the air–water interface remains unclear. To better understand this process, we have employed multiple techniques on three model proteins, HSA, HEWL and IgG2. These proteins have diverse
304
M. Jayaraman et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 299–309
Fig. 5. Reversibility of HSA (A and D), HEWL (B and E) and IgG2 (C and F) particles formed during agitation on 5 fold dilution either in 200 mM sodium phosphate buffer (pH 7.0) containing 50 mM NaCl (A–C) or in 10 mM sodium acetate formulation buffer (pH 5.5) (D–F).
structural and physical properties (Molecular weight, pI, surface hydrophobicity and thermal stability). Moreover, these model proteins have been previously investigated for their aggregation behavior under agitation and other stress conditions [24–26].
4.1. Mass balance during agitation All the model proteins lost a significant amount of monomer with formation of soluble aggregates (HSA and HEWL only) and
305
M. Jayaraman et al. / European Journal of Pharmaceutics and Biopharmaceutics 87 (2014) 299–309 Table 1 Average fluorescence intensities from DSF melting curves of the unagitated and 7-h agitated samples of HSA, HEWL and IgG2. Protein
Agitation time (h)
Average (three samples) ± SD
HSA
0 7
4470 ± 112 5493 ± 260
HEWL
0 7
2023 ± 4 2018 ± 3
IgG2
0 7
2071 ± 9 2069 ± 14
subvisible particles upon agitation. When the percentage of remaining protein monomers is compared with the sum of soluble aggregates by SEC-HPLC and insoluble proteins converted into subvisible particles detected by HIAC, it is clear that the total number does not add up to 100%. The higher percentage of monomer loss than what can be accounted for by SEC and HIAC could be attributed to several possibilities. First, particles of submicron range (128 nm aggregates immediately after agitation. One, however, has to
understand that these size estimations as measured by DLS may not be very accurate, as the DLS method cannot distinguish well protein monomers from dimers and trimers in solution and is semi-quantitative in estimating higher molecular weight aggregates [32,33]. Nevertheless, the lack of smaller oligomeric aggregates (
