Thank You - How to Measure the Shear Thinning Viscosity of mAb with 100 μL for Accurate Estimation of Injection Force

Key Words:   Viscosity, Concentration, Protein Therapeutics, mAb, Shear Rate Sweep, Injectability, Small Volume Viscometer

Goal:  The goal of this application brief is to present a method that allows viscosity measurement of high concentration mAb over a wide range of shear rates for volumes as low as 100 μL with m-VROC®II. Shear rate dependent viscosity measurement is required to estimate accurate injection force of shear thinning mAb formulations.

Introduction

It is now generally accepted that high concentration mAb solutions are non-Newtonian shear thinning liquids for which viscosity decreases with shear rate. Because of the shear thinning behavior, shear rate dependence of the viscosity from low shear rates up to the injection shear rates in the needle is required in order to estimate accurate injection force. We have developed and present a method that enables the measurement of viscosity for a wide range of shear rates for volumes as small as 100 μL with the m-VROC II viscometer.

Experimental

Sample preparation

Bovine γ-Globulin (BγG) solutions were prepared as a model mAb solution at four different concentrations (100, 150, 200, and 250 mg/mL). To minimize errors, a stock solution of 250 mg/mL was prepared by mixing BγG powder with PBS buffer overnight in a slow mixer. All the lower concentrations were prepared by diluting the stock solution. For each concentration, 100 μL was used for the measurement.

Test

1. Sample Loading: For each concentration, ~100 µL of sample was first aspirated into a positive displacement pipette (Gilson M100). Then the sample was injected into the backside of the 100 μL syringe. Please see the m-VROC II User’s Manual for more details on sample loading procedures.   Note that the sample must be free of air bubbles. Also make sure that the threaded area of the syringe is free of particles or fibers.
   
   
2. Prepare B05 and E02 chips for the measurement. If the viscosity of the sample is higher than 80 cP, the E05 chip is preferred to E02. Two chips are needed to measure viscosity at a wide shear rate range.
   
   
3. Firstly, viscosity of the sample is measured with E02 chip (maximum full scale pressure = 1,800 kPa, depth = 20 μm). The chip was connected to the loaded syringe and viscosity was measured at different shear rates with a level generated measurement protocol ([RS]LvlGen_12 Lvls_E02_100uL_Regular_v1). The measurement protocol is designed with 12 levels to measure viscosity automatically at different shear rates. After the measurement was finished, the sample was automatically recovered back to the syringe. Yield of the sample recovery is typically greater than 70% so that more than 70 μL is available for the next measurement. The recovered sample was typically loaded with small air bubbles near the tip of the syringe. These small air bubbles were first pushed out of the syringe for the next measurement.
   
   
4. Secondly, viscosity of the same sample is measured with B05 chip (full scale pressure = 40 kPa, depth = 50 μm). The syringe with recovered sample was connected to a B05 chip for the second shear rate sweep measurement with a level generated measurement protocol ([RS]LvlGen_12 Lvls_B05_100uL_Regular_ v1). This protocol is designed to measure viscosity at different shear rates automatically. The measured shear rates with the B05 chip will be lower than those with the E02 chip. Therefore, measurements with two chips expand the range of shear rates as shown in Figure 1. After the measurement was finished, each chip was cleaned with a buffer cleaning protocol on the Chip Cleaning Station (CCS).

Results

Figure 1 shows the viscosity as a function of shear rate for four BγG solutions in PBS buffer, as measured with the B05 and E02 chips. As shown, BγG solutions exhibit non-Newtonian shear thinning behavior. For the samples that exhibited non-Newtonian behavior, the WRM correction was applied to the shear-thinning region to determine the true shear rate and viscosity with the data analysis program Clariti^TM. Each data point is an average of five repeated measurements. All error bars correspond in length to three times the standard deviation and are smaller than the size of the symbols, demonstrating high reproducibility of our viscosity measurements.

Samples with concentration 200 mg/mL and above have a Newtonian plateau followed by a shear thinning regime. The critical shear rate (γ_𝑐) indicating the onset of decrease of the viscosity shifts to lower shear rate as the plateau viscosity increases. For example, the low concentrated solution (C_BγG = 200 mg/mL) shows the onset at ~28,000 sec-1 whereas the high concentration solution (C_BγG = 250 mg/mL) shows the onset at ~5,000 sec-1. 

Figure 1: True viscosity vs. shear rate for ten BγG concentrations. Error bars correspond to 3 standard deviations (3σ).

From the shear rate dependent viscosity data, one can estimate accurate injection force if injection rate and syringe geometries are given. In Table 1, syringe geometries are listed.

Table 1. Potential syringe geometries. L is the syringe needle length, R_p the inner radius of the syringe barrel or piston, and R_n the inner radius of the needle for different needle gauges.

Table 2 lists the calculated injection force (IF) for the four BγG samples for the needle gauges from 22 to 28 and for an injection rate of 0.4 mL/s. The estimation of the injection force was made using a rigorous formula with a few assumptions. If the maximum shear rate is not measured, then shear stress is extrapolated using a quadratic relationship with the shear rate. Contribution of the flow in the syringe barrel to the injection force is neglected. The detailed injection force calculation and the role of high shear rate viscosity on accurate estimation of injection force can be found in our April 2023 webinar Predicting Injectability: The Role of High Shear Rate Viscosity Measurements). In addition, the estimation calculator of injection force for shear thinning solutions is available in our data analysis program Clariti. 

Table 2- Estimated injection force (IF) for non-Newtonian mAb formulations. The injection force for each needle was calculated for the dimensions listed in Table 1, and the injection rate of 0.4 mL/s.

Concluding Remarks

In this note we presented a method to make reliable and accurate viscosity measurements over a wide range of shear rates for limited sample volume of mAb solutions with m-VROC II. Our results demonstrated that shear-rate dependent viscosity is accurately measured, thus enabling an accurate estimation of the injection force of shear thinning mAb solutions.

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