Intrinsic Viscosity Measurement for Optimal Therapeutic Formulation
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1 Intinsic Viscosity Measuement fo Optimal Theapeutic Fomulation Optimization of candidate monoclonal antibodies (mab) is an essential step in the development of theapeutic fomulations. Injectability is a key popety equied to assess the safety and efficacy of candidate mab s. Viscosity of a fomulation is the sole paamete that dictates injectability and is thus citically impotant to chaacteize duing the development pocess. In this bief, we demonstate a method fo accuate and epeatable viscosity measuement that eliably pedicts injectability and povides impotant insights about inte-molecula inteactions involving candidate compounds. Automated measuement of viscosity with VRO initium can incease the thoughput and efficiency of the fomulation development pipeline, optimizing use of valuable time and esouces. Mateial: Bovine γ-globulin fom Sigma Aldich (Lot # SLBO4091V) was used as a model mab. 10 mm Phosphate Buffeed Saline was pepaed by dissolving dy powde fom Sigma Aldich (Lot 039K8200) in one lite of deionized wate. Bovine seum albumin (BSA) obtained fom Sigma Aldich (Lot 060M1708V) was also used fo this study. Sucose was puchased fom Fishe Scientific (Lot ). Sample Pepaation: Thee media solutions wee pepaed fo this study: PBS buffe, 4 wt% sucose solution in deionized wate, and 30 mg/ml BSA solution in PBS. Viscosity of each media at 25 o (as measued with initium) is shown in the table below. Table 1. Measued viscosities of thee media. Media PBS buffe 30 mg/ml BSA in PBS 4 wt% Sucose solution in PBS Viscosity, mpa-s % RSD RSD (elative standad deviation) was calculated fom 8 data points. See below fo measuement. Measuement: VRO initium was used to test γ-globulin solutions in vaious media using the same measuement potocol, which is as follows: 12 measuements each sample at 600 µl/min (shea ate = 11,786 s -1 ) at 25 o, with sample etieval between each measuement. The fist fou data points fom each measuement wee discaded because of possible incopoation of small bubbles due to ai entapment. Sample volume was 100 µl in each case. The measuement potocol also woks well with 50 µl sample volumes. ontact RheoSense fo moe infomation. Fo all measuements, %RSD was less than 0.5%. Results: Measued viscosities of γ-globulin solutions in the thee diffeent media ae summaized in Tables 2 4 below. Table 2. Viscosities of γ-globulin solutions in PBS buffe
2 , mg/ml η, mpa-s %RSD η η sp/ φ (vol. faction) In the table, η, η sp/, and φ ae calculated using the following equations: η( solution) η = ; η( solvent) η = η sp 1 4π ; and φ = R 3 3 h N M a Whee R h is the hydodynamic adius, is the concentation, N a is Avogado s numbe, and M is the molecula weight. The hydodynamic adius is calculated fom the intinsic viscosity [η] using the following equation fo an appaent spheical shape: R h 3[ η] M = 3 10 πn a The intinsic viscosity is calculated by extapolating η sp/c to zeo concentation as shown in Figue 1. η sp / , mg/ml Figue 1. η sp/ vs. plot to obtain [η] and K H. (see equation below fo the definition of [η] and K H.
3 Tables 3 and 4 summaize the analysis of the viscosity data of γ-globulin solutions in 30 mg/ml BSA and 4 wt% sucose in PBS media. Table 3. Analysis of the viscosity data of γ-globulin in 30 mg/ml BSA in PBS buffe., mg/ml η, mpa-s %RSD η η sp/ φ (vol. faction) Table 4. Analysis of the viscosity data of γ-globulin in 4 wt% sucose in PBS buffe., mg/ml η, mpa-s %RSD η η sp/ φ (vol. faction) Figue 2 compaes the change of η sp/ with, which yields [η] and K H fo the γ-globulin solution in the thee media. As shown in Table 5, γ-globulin molecules inteact with the media diffeently. The intinsic viscosity is smallest and K H is the lagest in 4 wt% sucose in PBS medium. The esults suggest that γ- Globulin molecules ae the smallest in 4% sucose in PBS media in dilute solution. Howeve, highe K H[η] 2 suggests that viscosity of g-globulin solution in 30 mg/ml BSA in PBS will incease faste as concentation inceases since η follows the equation below at low concentations. η = [ η] K H [ η]
4 η sp / PBS 30 mg/ml BSA in PBS 4% Sucose in PBS , mg/ml Figue 2. η sp/ of γ-globulin solutions in vaious media. Table 5 summaizes the calculated intinsic viscosities, hydodynamic adii, and Huggins constants of γ- Globulin in thee diffeent media. PBS 30 mg/ml BSA in PBS 4 wt% Sucose in PBS [η], ml/mg R h, nm K H η φ PBS 30 mg/ml BSA in PBS 4% Sucose in PBS Batchelo's
5 Figue 3. η of the γ-globulin solution in vaious media as a function of φ. In Figue 3, the elative viscosities, η, of the g-globulin solutions in vaious media ae plotted against the volume faction and compaed to the Batchelo s calculation of the viscosity of suspensions of had sphees with hydodynamic inteaction 1. As shown in the figue, the elative viscosity of γ-globulin deviates fom the had sphee pediction at a volume faction of Also noted is that the viscosity incease elative to volume faction is most apid in 4 wt% sucose in PBS media. onclusions: 1. Viscosity measuement is used fo the intinsic viscosity and Huggins constant. 2. The intinsic viscosity povides hydodynamic adius, which indicates the inteaction of the potein molecules with media. 3. Molecula size can be calculated fom the intinsic viscosity measuement even if the media contains othe potein molecules. 4. The combination of [η] and K H can be useful when optimizing fomulation fo stability, efficacy, and injectability. The value of K H[η] 2 can be used to pedict how fast the viscosity inceases with concentation, which is vey useful infomation. 1 Batchelo, G. K. (1977). The effect of Bownian motion on the bulk stess in a suspension of spheical paticles. J. Fluid Mech. 83, If you have questions egading the measuement o need moe infomation about VRO initium o othe applications, please contact us: Main Office Infomation info@ Sales Sales@
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