Sample vials on the autosampler. Images: MalvernAs interest and investment in biopharmaceuticals grows, the pressure to innovate and rapidly deliver new therapies increases. While many avenues may be pursued, the high cost of developing biological molecules increases the need to advance only those therapies with the greatest likelihood of becoming manufacturable, efficacious, safe and profitable products. Therefore, developers are driven to select the “right” candidate molecules as early as possible in development in order to “de-risk” further investment.

Making that candidate selection involves a number of physicochemical testing processes. These are designed to eliminate molecules that might experience formulation, delivery or production problems downstream. Appropriate testing methods are only now beginning to emerge, and the push is for high-throughput automated analysis that can cope with very small volumes, and ideally is non-destructive. This article explores a new approach to viscosity measurement that is especially well suited to pre-formulation and formulation development.

Decision-making parameters
Post discovery, properties such as viscosity and the physical and chemical stability of a formulation can be deciding factors in whether a biological molecule attracts ongoing interest and resources. However, at the initial stage of development only very small sample volumes are available upon which to perform a whole battery of testing. Early measurement of viscosity under likely formulation conditions is especially important as the final product is most likely delivered by injection, in small volumes at high concentration. For biological molecules, high concentration very often also means high viscosity, with implications for downstream development.

A novel solution to the challenge of rapidly and non-destructively measuring viscosity on multiple low-volume samples brings together the techniques of ultraviolet (UV) area imaging and microcapillary viscometry.

Combining technologies for accurate results on low sample volumes

Dual pass microcapillary viscometry
A sample’s viscosity can be determined from the time it takes to travel between two points—relative to a reference of known viscosity—at constant pressure and temperature. Capillary viscometry is an established technique that uses this principle, relying fundamentally on measuring the transit time of a sample along a capillary, from the site of its injection to the point where it reaches a suitable detector. However, a significant drawback in traditional implementations of this technique is its proneness to injection time errors. These errors can adversely affect subsequent viscosity calculations to a significant degree, calling into question the accuracy and robustness of the information delivered.  

One approach to overcoming the injection time dependency of capillary viscometry is the development of a dual-pass microcapillary system. This allows sample detection at two windows along a microcapillary with transit time between the windows measured with great precision. This figure is then used in all calculations, completely removing any reliance on sample injection time.


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Viscosity measurements of BSA in histidine buffer with and without arginine.

UV area imaging
Coupled with the dual-pass microcapillary system is the use of highly sensitive UV area imaging for sample detection. UV area imaging at the two windows monitors the time-dependent UV-absorbance profiles of samples as they migrate through the microcapillary. Specific wavelength selection enables its use for the analysis of proteins and peptides, and indeed any other UV chromophore-containing molecules.

The UV imaging array generates a series of individual snapshots targeted at the species-specific absorbance profile. Once measurements are made, signal processing functions determine sample viscosity and use an appropriate time displacement to combine and average the UV snapshots. These data are then converted to give an accurate measurement of viscosity. Separate measurements of molecular size and concentration can also be made.

Since measurement is not dependent on the physical characteristics of a sample, but relies instead on the unique UV absorbance profile of the molecule, the method works with very low sample volumes. These can be below 10 uL for viscosity and as little as 10 nL for size. In addition, because UV absorbance is a property unique to individual chemical species, the signal response of the target molecule can be measured without interference from surrounding materials, allowing measurement even within complex matrices.

In-use demonstration
To illustrate viscosity measurement using the combined technologies, dilutions made from stock solutions of bovine serum albumin in two different formulation buffers—one containing arginine and one without—were analyzed using the commercially available UV area imaging/dual pass microcapillary system. Vials containing 100 µL aliquots were placed on the system’s autosampler carousel (Fig 1) which feeds the microcapillary. The reference sample used was pure water spiked with caffeine.

Equation 1 shows the relationship between the time taken for the sample (Δt) and reference (Δt0) fronts to pass between the two detection windows, and the sample-specific viscosity (ηsp).

Equation 1



L is the total capillary length, and l1 and l2 are the lengths measured to the first and second detection windows in the capillary. All data acquisition and processing is under the control of the instrument software and viscosity for each formulation was determined using the relationship described above. Frontal analyses of viscosity traces (Figure 2A) were used to obtain Δt and Δt0 for the sample and for the viscosity reference (water). The remaining parameters Ll1 and lare known from the dimensions of the microcapillary.

Figure 2A is a screen capture showing sample fronts at Windows 1 and 2, and corresponding Δt values for the two different BSA formulations. Figure 2B is a plot of absolute viscosity as a function of BSA concentration in the two different formulations. At concentrations higher than 250 mg/mL, the formulations containing arginine have lower viscosities than those without, consistent with the documented viscosity-lowering properties of arginine. The dashed line is a typical viscosity screening threshold for acceptable candidate selection. Four of the results fall above this threshold (20 cP) and would likely be excluded from further development.

Screening potential
The results illustrate that the combination of UV area imaging and dual pass microcapillary viscometry can discern differences between the viscosities of different protein formulations. Being able to do this rapidly and reliably has value in screening appropriate formulation candidates, potentially against viscosity thresholds. Integrating these two technologies into a single system has produced a new automated tool with significant