What biophysical attributes of biopharmaceuticals are measured, and why?

During the development of new biopharmaceuticals, a range of attributes must be assessed. Here we look at the specifics of these tests – what characteristics are measured, and why they are important.


Biopharmaceuticals is a broad term encompassing proteins, antibodies, hormones, enzymes and other biologically based therapeutics. During development of these complex molecules, a variety of characteristics must be thoroughly examined, to ensure the safety and efficacy of the final formulation.



aggregation icon

From dimerization to large visible aggregates, formed by strong or weak bonds, in ordered or disordered arrays – aggregation has a major impact on biopharmaceuticals. Aggregation of biopharmaceuticals can be triggered by a range of different factors -the formulation and additives, temperature, pH or ionic strength can all play a part.

The effects of aggregates can range from reduced binding affinity, to immunogenicity and serious health impacts. The formation of aggregates in epoetin-α after a formulation change is a well-documented example, which triggered cases of Pure Red Cell Aplasia in patients during the 1990s [1].

Zhang et al demonstrated that MDS can be used to track the fibril formation of α-synuclein [2].



The modification of Immunoglobulin G (IgG) molecules can have an impact on safety as well as efficacy. Standard glycosylation via an N-linked glycan at residue 297 in the CH2 domain can change the binding to some Fc receptors. Less common O-linked glycosylation tends to occur on unstructured regions and can produce less predictable results. As such this form is less commonly used.

As changes in glycosylation can influence pharmacodynamics and pharmacokinetics, it is considered a critical quality attribute [3]. Galactosylation has been shown to affect IgG1 conformation, promoting activation of the complement system [4].


Other post-translational modifications

Aside from glycosylation, other modifications can be carried out on biopharmaceuticals. Acetylation or succinimylation at exposed lysine residues or N-terminal amine groups can cause heterogeneity. An N-terminal glutamine can be converted to a pyroglutamate, changing the charge and stability of the molecule. Oxidation of methionine can change stability, and the deamidation of asparagine residues to aspartate gives a product prone to isomerization. Volume increasing agents such as PEG can be utilized (“PEGylation”) to reduce renal filtration clearance.

Any combination of these changes can impact the binding and stability of the product, as well as formulation development.

Conducting an assessment such as those demonstrated by Herling et al [5], before and after modifications could indicate to what extent the changes made have impacted binding.


Non-specific binding

non specific binding icon

Although biopharmaceuticals are by nature highly specific, they may show low affinity binding with other species. If this binding is with an abundant species, it can form a sink reducing the amount of on-target binding.

This is normally identified in early in vivo testing, but can be tested through some screening techniques before this stage.


FcR binding

IgGs with an intact Fc region will exhibit binding to various Fc receptors – including the surface of immune and endothelial cells. These receptors can have inhibitory, activatory or metabolic effects.

Using antibody fragments can avoid this effect, as the Fc region is removed.


Thermal stability

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Differential Scanning Calorimetry (DSC) is used to measure the temperature(s) that unfolding occurs at. Although broadly related to overall stability, some of these changes may be due to conformational flexibility of the molecule.

Knowledge on this point can help to inform storage guidelines, ensuring the product remains safe and stable between production and use.


Surface charge

The surface charge of a protein can change over manufacturing and purification. Charge is determined by 5 key amino acids; basic lysine and arginine, acidic aspartate and glutamate and zwitterionic histidine.

Changes in surface charge have been shown to affect binding affinities, and in turn biological response [6], as well as overall stability of the molecule [7].


Buffer compatibility

buffer compatability icon

The buffer a biopharmaceutical is stored in can affect aggregation, stability, charge, activity and more.

The behavior and form a molecule takes in different buffers is therefore investigated to ensure maximum stability.

A fast check on samples in different buffers can help in assessing this. MDS is ideal as it only requires a small sample volume and gives results in minutes.



Soluble proteins are usually formed in such a way that the hydrophobic residues are in the core, contributing to stability, with few on the surface (with the exception of some binding regions).

As hydrophobic regions are linked to aggregation, knowledge on the molecule’s structure in this aspect can contribute to formulation development and concentration limits.



heterogeneity icon

All of the parameters discussed here can contribute heterogeneity. Although proteins are intrinsically heterogenous, for the purpose of therapeutics this should be minimized and consistent.

No analytical technique is capable of giving a complete picture in this regard, instead results from a range of methods need to be considered simultaneously to give a full picture.

It is clear that the biophysical characteristics of biopharmaceuticals can have a profound effect on their stability, structure, binding, efficacy and safety. This is why robust and reliable analytical techniques are central to research and development in this field.

To learn more about the various techniques available for protein size or quantification testing, see our previous posts. Alternatively contact us here if you have any further questions about biophysical analysis in biopharmaceutical development and manufacturing.



  1. Bennett et al, New England Journal of Medicine, 2004 Sept 30; (351):1403-1408
  2. Zhang et al, Chembiochem, 2016 Oct 17;17(20):1920-1924
  3. Dietmar et al, Glycobiology, 2015 Dec 1;25(12):1325-1334
  4. Houde et al, Mol Cell Proteomics, 2010 Aug;9(8):1716-1728
  5. Herling et al, Biophys J. 2016 May 10; 110(9): 1957–1966.
  6. Hintersteiner et al, Biotechnol J, 2016 Dec; 11(12):1617-1627
  7. Strickler et al, Biochemistry, 2006 Feb 7;45(9):2791-2766