0 Rates

The viscosity of protein therapeutics


Proteins are biological macromolecules composed of amino acids. When many single amino acids are linked together, a polypeptide is formed. One or more polypeptide chains twisted into a 3D shape form a protein. There are 20 different amino acids which are combined to build a variety of proteins. The sequence (order) of the amino acids determines the 3D structure and the final function of the protein (Figure 1).[1]

Figure 1. The amino acid alanine, the peptide (chain of various amino acids) glutathione, and the protein (3D structure of various peptides) human serum albumin (http://www.rcsb.org/structure/1AO6, Sugio, S. et al., 1998)

Protein engineering

Protein engineering focuses on the design of new proteins or enzymes with new or desired functions. General targets of protein engineering are the modification of enzyme stability, activity, substrate specificity and enantioselectivity. The controlled manipulation of proteins allows a better understanding of functionality and enables further improvements in protein properties. [2]

The three main strategies of protein modification are: directed evolution, rational design, and a combination of both, semi-rational design. The directed evolution method uses a controlled environment to induce mutations and selection, while rational design manipulates amino acids directly.[3]

A broad range of applications can be covered with modified proteins or enzymes, including biocatalysis for food as well as environmental, medical and nanobiotechnology applications. [4] Some examples of modified enzymes for use in the food, detergent and paper industries are proteases and amylases. Other enzymes like peroxidases and oxygenases are used in the environmental sector.[5]

Because of the central role of proteins in biological functions, protein engineering is a crucial technology for new biological therapies. As a consequence, therapeutic proteins show great potential for the targeted treatment of diseases such as cancer, Alzheimer's, Parkinson's and HIV.[6]

Advantages of protein/enzyme engineering in comparison to traditional chemical reactions:

  • Efficiency (in enhancing the rate of chemical reactions)
  • Substrate specificity (ability to discriminate between potential substrates)
  • Environmental friendliness (no organic solvent or heavy metal toxic waste)
  • Cheap and easy usage (many enzymes are commercially available)
  • Possibility of enzyme combination (can be used together in sequence or cooperatively to catalyze multistep reactions)
  • Regio- and stereospecific reactions (produce chiral pure products)
  • Suitability for many different applications (medicine, chemical industry, food processing, agriculture)


Therapeutic protein definition

The use of proteins as therapeutics has attracted more and more interest since the invention of the first protein therapeutic, human insulin, for the treatment of diabetes.[7]

The success of protein therapeutics in the treatment of various diseases is based on their many advantages over small-molecule drugs.

  • Proteins typically exhibit highly specific and complex functions which cannot be mimicked by small-molecule drugs in the body.
  • Proteins can be engineered to show high affinity for their target and therefore do not interfere with other biological processes in the body.
  • Many proteins used as therapeutics are produced by the body naturally. Therefore, they are often well-tolerated and do not cause immune responses.
  • Clinical and approval times are much shorter than for small-molecule drugs.

Additionally, the ongoing development of new techniques in protein engineering enables the formulation of new therapeutics specifically designed to target certain diseases.[8]

Apart from the many benefits of protein therapeutics, there are still some major problems. These complications include rapid degradation and excretion by patients, which requires repeated administration. This increases in turn the chances for an immunological response and raises therapy costs. The main strategy to overcome these limitations is PEGylation, where the protein is linked to polyethylene glycol to improve properties and reduce immunogenicity and proteolytic cleavage.[5] 

Types of therapeutic proteins

According to their functions and therapeutic applications, protein therapeutics can be classified into four groups. Proteins with enzymatic or regulatory activity, special target proteins, and vaccines are further divided into individual categories. Group 1 and 2 have the advantage of approval by the FDA (U.S. Food and Drug Administration), while group 3 and 4 are investigated with in vivo/in vitro experiments.[9]

In addition to classification according to their pharmacological activities, grouping according to molecule types (antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics) and molecular mechanisms (binding non-covalently to target, e.g. mAbs; affecting covalent bonds, e.g., enzymes; and exerting activity without specific interactions, e.g., serum albumin) is also possible.[10]

Group 1: Protein therapeutics with enzymatic or regulatory activity

  • 1a: Replacement of a protein that is deficient or abnormal.
  • 1b: Augmenting an existing pathway.
  • 1c: Providing a novel function or activity.

Group 2: Protein therapeutics with special targeting

  • 2a: Interfering with a molecule or organism.
  • 2b: Delivering other compounds or proteins.

Group 3: Protein vaccines

  • 3a: Protecting against a deleterious foreign agent.
  • 3b: Treating an autoimmune disease.
  • 3c: Treating cancer.

Group 4: Proteins used for diagnostics

The largest and best-selling therapeutic protein group on the market is currently the first one, whereas the fastest growing therapeutic proteins are the monoclonal antibodies. [10]

Therapeutic protein examples

Most protein therapeutics are created by recombinant DNA and are used for medical applications. Therapeutic proteins have been shown to have significant benefits for the health system, serving as agents for the treatment of various diseases.[9] Some of the most important therapeutic proteins, their potential applications, and classifications are summarized in Table 2.

Table 2. Examples of proteins used in therapeutics and diagnosis including their classifications.

Protein therapeutic Classification Function Clinical use
Insulin Group 1 Regulates blood glucose Diabetes mellitus
Hyaluronidase Group 1 Increases tissue permeability, allows faster drug absorption Anesthetic in ophthalmic surgery
Botulinum toxin Group 1 Causes paralysis Movement disorders, cosmetic issues
Trastuzumab Group 2 mAb that controls cancer cell growth Breast cancer
Hepatitis B antigen Group 3 Non-infectious protein on surface of hepatitis B virus Hepatitis B vaccination
OspA Group 3 Non-infectious protein on surface of Borrelia burgdorferi Lyme disease vaccination
HIV antigens Group 4 Immunoassay that detects human antibodies to HIV Diagnosis of HIV infection

Monoclonal antibodies (mAbs)

Antibodies are a special group of proteins. When external substances that are foreign to the body (antigens like viruses, bacteria, or allergens) enter the body, our immune system recognizes these substances and produces antibodies. These antibodies can bind to antigens and inactivate them by building an immune complex. These complexes are then transported to the liver and removed from the body. 

Figure 2 Antibody-antigen interaction mechanism

In contrast to the antibodies produced by our body, monoclonal antibodies (mAbs) cannot be isolated from humans, but have to be produced in the lab according to certain techniques and are far more specific. Research on and use of mAbs have significantly risen in recent years, and they have become one of the predominant forms of protein-based therapeutics.[7] Due to their specific design, they are very efficient and used in treatments for many diseases like Alzheimer’s, multiple sclerosis and various forms of cancer. mAbs can also be used as transporters for other drugs in cancer therapy, thereby limiting the toxicity of the transported drug while increasing its efficiency.[8] They function as follows: The drug is linked to the mAb, forming an antibody-drug conjugate, and is transported directly to the target (cancer cell). The cancer cell absorbs the drug-mAb combination and is killed.[11]

Protein therapeutics – Impact of viscosity

Therapeutic protein solutions are very complex systems due to the proteins’ structure and large size. The manufacturing and production of these protein solutions are also complex. On the one hand, the synthesis of the proteins themselves does not follow simple chemical pathways as is the case for small-molecule drugs, and on the other hand, their production may include thousands of critical process steps.[3] Additionally, the synthesized proteins have to be stable in both solution and during administration in order to maintain drug efficiency and patient safety. The influence of the solution’s viscosity and stability is highly significant and affects the applicable administration route of protein therapeutics. These topics will be discussed in the following chapters.

Concentration-dependent viscosity behavior

Traditionally, protein therapeutics have been formulated at rather low concentrations (e.g. 20 mg/mL) for intravenous administration in hospitals.[7] However, other administration routes, such as subcutaneous injections, become more and more desirable due to the convenience of self-administration.[12] Furthermore, patients suffering from chronic diseases who require frequent dosing would greatly benefit from alternative administration routes.[13] Unfortunately, subcutaneous injections are limited to small injection volumes (~1.5 mL). In comparison to intravenous administration, where large volumes can be applied, the protein concentration of the solutions used for subcutaneous injections has to be increased to enable sufficient treatment. Depending on the protein concentration, several 100 mg/mL are required to achieve the same effect when compared to intravenous administration.[14]

For protein therapeutics with a small molecular weight like insulin, subcutaneous injections are already common practice. However, insulin is a rather small protein in comparison to other proteins like mAbs (Figure 3). The larger size and the more complex structure of mAbs have a huge impact on the solution’s properties, especially on its viscosity, which is a limiting factor for administration forms like subcutaneous injections.

Figure 3. Comparison of the size of the small molecule drug Ibuprofen (206 Da molecular weight), insulin (5.8 kDa molecular weight, www.rcsb.org/structure/3I40, Timofeev, V.I. et al. 2010) and Trastuzumab an mAb for treating cancer (140 kDa molecular weight, www.rcsb.org/structure/4HJG, Donaldson J:M., 2013)

Above a certain concentration (protein-dependent), the viscosity of the protein solutions may increase dramatically. However, not every protein shows the same concentration-dependent viscosity increase.[15]  

Figure 4. Concentration-dependent viscosity behavior of two mAbs in solution (adapted from [6])

The extent of the viscosity changes with increasing protein concentration, and depends on several factors. Basically, it can be said that the larger the protein, the higher the concentration-dependent increase of the solution’s viscosity. Apart from size, the interactions between the separate proteins, called protein-protein interactions or PPIs, have a major influence on the solution’s viscosity.[16] The higher the protein concentration in the solution gets, the smaller the distances between the separate molecules become. This leads to an increase of PPIs, potentially resulting in high viscosities and stability problems like aggregation.[17]  

Stability of highly viscous protein solutions

The effectiveness of protein therapeutics is largely dependent on the protein being present in its active form, i.e. its native structure. Only in its native form can it fulfill its full biological function. If proteins aggregate (clump together) or denature (3D structure is lost) in solution, the biological function of the protein, and therefore the efficiency of the protein therapeutic, is also lost. Additionally, patient safety cannot be guaranteed anymore. 

Figure 5. Protein in its native and denatured form and.

For these reasons, a key property of protein therapeutics is their stability. Guaranteeing drug stability is challenging, especially when working with highly concentrated protein solutions. In dilute solutions, the stability is only dependent on PPIs such as long-range interactions, whereas in highly concentrated solutions, the density of proteins of the same volume is much bigger, and other PPIs (short-range interactions) also start to influence the stability.[14] The amino acid sequence and structure of the protein influence the strength of PPIs, as well as solution conditions like pH, ionic strength, and the presence of other compounds.[17] Protein solutions are by nature prone to stability problems such as precipitation, hazing, denaturing, gel formation, self-association, and aggregation caused by changes in temperature, shear, solvent composition, containers used, and others.[18]

To control and investigate under which conditions a solution remains stable, viscosity studies described in chapter 4.4 are used. 

Administration of highly viscous protein solutions

Aside from drug stability, high viscosities resulting from high protein concentrations pose a challenge for subcutaneous injection where low-viscosity solutions are desired (<50 mPa.s). A solution’s viscosity influences the injectability of a substance. Larger needle gauges or higher injection forces are needed for the injection of highly viscous solutions resulting in a lower patient tolerance due to injection pain.[19] The functioning of auto-injection devices is also affected by the properties of high-viscosity solutions. The design and the material used for the devices must be able to bear the force needed for injections. Problems like breakage and malfunctions in the products can occur. A common practice to reduce the injection force is a lower injection rate. However, the patients’ tolerance is rather low regarding long injection times.[12]

Apart from injectability and injection-device manufacturing, the high viscosity of protein solutions also affects the processing of these drugs. In filtration steps, for instance, high viscosities can lead to increased backpressure in the pumps which increases processing time and manufacturing costs. In this case, product loss is stated as well.[20]

Analyzing proteins and influencing the viscosity of highly concentrated protein solutions

It is highly important to analyze proteins before preparing therapeutics. To determine the molecular weight for characterizing proteins and distinguishing between proteins of different size, techniques such as size exclusion chromatography (SEC)[17], dynamic light scattering[21], small angle x-ray scattering[22], and dilute solution viscometry[21] are used.

Much research has been done in controlling and investigating the concentration-dependent viscosity behavior for protein therapeutics. Viscosity studies are used to test how the viscosity of protein solutions can be influenced and manipulated while maintaining the protein solution’s effectiveness and stability for a long shelf life.[14] To determine the solution stability and changes in viscosity, techniques such as dynamic light scattering (DLS)[16][17], rheological measurements[14][16][17], glass capillary viscometers[14][15], and rolling ball viscometers[14] are used.

One approach in viscosity studies is to influence the viscosity of a protein solution in such a way that PPIs are decreased and aggregation is hindered, resulting in a lower solution viscosity. Therefore, parameters like pH, temperature, solvent, etc. are changed, and the solution viscosity is monitored before and after the changes, using model compounds like serum albumin or lysozyme[14][16][23] Another approach is to search for additives like amino acids and salts which influence the PPIs and therefore lead to a viscosity decrease and further enhance the solution stability.[18] Figure 6 shows an example of a study in which a salt (sodium chloride, NaCl) was added to serum albumin solution to investigate the change in dynamic viscosity.[16]

Figure 6. Viscosity of serum albumin in the presence (red dots) or absence (blue dots) of sodium chloride (adapted from [16]).


There is continuous interest in protein therapeutics for treating various diseases. It is to be expected that the efforts for finding new formulations and comfortable administration routes will further increase in the years to come. To meet these goals, understanding the viscosity-concentration dependency of new formulations will be essential. Equal importance should be given to the development of new additives and formulation conditions which decrease the viscosities of the final therapeutics, while maintaining stability and long shelf life without influences on the drugs’ effectiveness and the patients’ safety.

Application Reports


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