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European Biopharmaceutical Review

Ultra Culture

Of the many analytical challenges facing researchers during the early development of pharmaceuticals, the measurement of viscosity is especially important. Being able to accurately determine this can reduce the number of candidate failures during subsequent stages.

Measuring viscosity during the preformulation development of pharmaceuticals – particularly biopharmaceuticals – can be especially challenging. Researchers will be dealing with extremely small volumes of highly valuable samples, where measurement is needed under formulation conditions, often at high concentrations.

Conventional techniques for measuring viscosity struggle under these circumstances. Coupled with this is the increasing demand for automated methodologies which can perform high throughput measurements of more than one parameter of the same small sample. This article describes the application of ultraviolet (UV) area imaging to automated viscosity and molecular size measurements for biological formulations, and its uses and advantages over conventional techniques.

Injectable Biotherapeutics

In the development of biotherapeutics, drug efficacy and patient experience are crucial parameters. Since most biological molecules are unable to withstand the rigours of the gastrointestinal route, the usual method of administration is through intravenous, intramuscular or subcutaneous injection. Concentrations of the active molecules must be relatively high – typically more than 100mg/mL – in order to compensate for their short plasma half-lives.

One of the biggest challenges is the development of highconcentration formulations (for maximum effectiveness) that can be injected at small volume (for patient comfort). In biopharmaceuticals, high-concentration often means high viscosity and potential problems with parenteral delivery.

The formulation of biologics is an area of intense interest and one where understanding, and the analytical toolkit to support it, is developing fast. However, one measurement challenge that is only being fully addressed now is the ability to screen formulations for low-viscosity attributes or against defined viscosity thresholds, at an early stage in development. Traditional techniques for measuring viscosity – which include rotational rheometry and standard capillary viscometry – are ill-suited to the demands of early biopharmaceutical development, where multiple tests must be carried out on very small volumes of extremely high-value materials. Low volume analysis, high throughput and sample recovery are all priorities on the list of requirements.

A new solution is the combination of UV area imaging with microcapillary viscometry to deliver rapid, high throughput, non-destructive viscosity and molecular size measurements for biopharmaceutical formulations. Being able to measure viscosity reliably at an earlier stage in the development process opens up the possibility of selecting candidate biotherapeutic formulations with improved syringeability and injectability.

UV Area Imaging

In this application, UV area imaging is used to monitor the time-dependent absorbance profile of UV-active samples, such as proteins, peptides and other UV chromophore-containing molecules, as they migrate through a microcapillary. Capillary viscometry has long been used to determine viscosity from a simple measurement of time – from injection to detector – but has traditionally been subject to injection time errors that can result in significant mistakes in subsequent calculations.

In this new system, a dual-pass capillary design detects samples at two windows along the capillary, enabling precise measurement of the transit time between windows and of the sample’s viscosity (see Figure 1, page 24). Both viscosity and size analyses are based on time-based changes that occur between the two windows.

At a known constant pressure and temperature, the viscosity of a sample can be determined from the time it takes to travel between two points, relative to a reference sample of a known viscosity. Sample detection is achieved through a series of individual snapshots taken using the UV imaging array, targeted at the species-specific absorbance profile. A signal processing algorithm determines the sample’s viscosity and uses an appropriate time displacement to combine and average the snapshots. This is then converted into an accurate measurement of viscosity, molecular size and concentration.

Measurement relies on the unique absorbance profile of the target species rather than its physical characteristics. This means that it can work with low sample volumes of less than 10 microlitres for viscosity and less than 10 nanolitres for size.

Technique Comparison

As UV analysis is not a destructive technique; samples undergoing analysis are recoverable. This contrasts with conventional rheometry techniques which, while capable of measuring protein sample viscosity, induce destructive shear thinning effects on the sample. The problem is exacerbated by the open sample-to-air interface that leaves the sample susceptible to shear, protein adsorption and corruption.

A comparison of rotational rheometry with UV area imaging-based viscometry is shown in Figure 2 and indicates a clear difference between the two techniques in terms of their influence on the sample. The absence of shear thinning observed in the microcapillary analysis suggests that in rotational rheometry results may be affected by loose structure formation at the airsample interface.

The fully enclosed microcapillary of the UV area imaging-based system does not present an air-to-sample interface, preventing sample corruption or contamination during analysis and facilitating sample re-use. When combined with the potential for high throughput analysis, these attributes make it a technique eminently suited to early biotherapeutic screening.

Flexibility ofAnalysis Type

One of the appeals of UV area imaging is that it is capable of multiple measurements. Not restricted to viscosity analysis alone, the methodology can also be applied to the analysis of both molecular size and sample concentration.

Size characterisation of small molecules within a complex matrix is notoriously difficult because of masking by larger species. As a result, size measurement traditionally requires the use of high sample concentrations to achieve effective analysis. Sizing with UV area imaging is not limited by the size of the molecule and therefore presents a potential solution to this problem.

As UV absorbance is a property unique to individual species – provided that the molecule’s UV absorbance is detected – it is possible to measure the signal response of the target molecule without the signal becoming flooded with that of surrounding species.

Size is calculated in a similar fashion to viscosity. As the sample moves through the capillary, diffusion of particles or molecules causes broadening of the peak, but this broadening is also tempered by transverse diffusion. The smaller the molecules, the more rapidly transverse diffusion occurs, resulting in a narrow peak. When larger molecules are present, transverse diffusion occurs less rapidly, resulting in a broader peak. The change in peak width between the two windows is used to calculate the hydrodynamic radius of the molecules of interest (see Figure 3).

Finally, UV area imaging can also be used as a detection method for the concentration of a particular active ingredient. The Beer-Lambert law directly relates UV absorbance to the concentration of chromophores present, making it possible to determine the amount of a particular species present in the solution, in addition to its size. The application of the UV area imaging detector enables three defining measurements: the viscosity of the overall solution, the size of the molecules present, and their concentration.

Example Application

The development of highly concentrated, low-viscosity formulations is currently an area of focused research. An important strategy to achieve this is the addition of viscosity-lowering excipient species to the formulation. The addition of small molecule excipients – such as arginine, dimethyl sulfoxide and hydrophobic salts – has been shown to reduce the viscosity of highly concentrated protein solutions by inhibiting the formation of aggregate networks.

A recent study illustrates how UV area imaging-based microcapillary viscometry discerns the differences between the viscosities of different protein-excipient formulations, demonstrating how such technology can be implemented as a tool in the screening of appropriate formulation candidates against a defined viscosity threshold (1). The following data were generated using a commercially available UV area imaging-based system (see Figure 2, page 24).

Buffer Solutions

Stock solutions of 400mg/mL of the protein Bovine Serum Albumin (BSA) were prepared in two different excipient buffer solutions – both 30mM histidine, pH 5.3 – but one of these histidine buffers also contained 200mM arginine. From these stocks, a series of dilutions was prepared and the concentration of each solution was determined using standard UV spectroscopy methods. Aliquots of 100μL were transferred into small vials and placed into the instrument’s autosampler carousel at 20°C. The progress of the sample front was monitored between the two detection windows at 214nm. The resulting viscosities were then recorded and plotted against one another and against a pre-determined viscosity threshold (see Figure 4).

Figure 4 shows the plot of absolute viscosity as a function of BSA concentration. As expected, the formulation’s absolute viscosity rose with increasing concentration. Concentrations of below 250mg/mL showed no discernible difference between either buffer solution. However, concentrations measured above 250mg/mL showed that the BSA formulations in the 30mM histidine buffer without arginine has a higher viscosity than those formulations containing arginine. The viscosity lowering ability of arginine is well documented, particularly within studies on monoclonal antibodies (2-4).


The production of high-concentration, low-viscosity biotherapeutic formulations is one of the key challenges faced by the pharmaceutical industry today. A new technique that combines UV area imaging with microcapillary viscometry supports biopharmaceutical development by offering effective high throughput technology capable of the non-destructive analysis of very low volume, unmodified samples at formulation concentrations.

This provides an opportunity to screen therapeutic candidates at an early stage of development to ascertain their viscosity profiles and highlight any likely problems in downstream processing. The cost benefit of progressing (or rejecting) more thoroughly screened candidate molecules where viscosity behaviour can be more accurately predicted is high.


1. Effect of excipients on protein formulation viscosity, a study of low volume viscometry using the Viscosizer 200: Malvern Instruments application note, 2013. Visit: malvern/kbase.nsf/allbyno/KB003711/$file/MRK1934-01.pdf
2. Kamerzell TJ et al, Polar solvents decrease the viscosity of high concentration IgG1 solutions through hydrophobic solvation and interaction: formulation and biocompatibility considerations, J Pharm Sci 102(4): pp1,182-1,193, 2013
3. Guo Z et al, Structure-activity relationship for hydrophobic salts as viscosity-lowering excipients for concentrated solutions of monoclonal antibodies, Pharm Res 29 (11): pp3,102-3,109, 2012
4. Liu J et al, Reversible self-association increases the viscosity of a concentrated monoclonal antibody in aqueous solution, J Pharm Sci 94(5): pp1,928-1,940, 2005

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About the author
Lisa Newey-Keane
is Malvern Instruments’ Biopharmaceutical Product Manager. She gained a PhD in microbiology, pathology and protein biochemistry from the University of Birmingham. Lisa then moved to the product characterisation team at Lonza Biologics, specialising in the use of high performance liquid chromatography and mass spectrometry for the analysis of therapeutic antibodies, and transferred these skills to her later work for Shimadzu. Lisa has also worked within bioanalysis at Novozymes and most recently at Quotient Bioresearch Cambridgeshire.
Lisa Newey-Keane
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