Monoclonal Antibody-drug Stability Optimization

Monoclonal antibody drugs are an important branch of biopharmaceuticals, which have received widespread attention due to their high targeting and specificity. With the continuous development of monoclonal antibody technology, antibody drugs play a pivotal role in disease prevention, diagnosis and treatment.

Antibody drugs, as a natural property of synthetic proteins, are susceptible to physical and chemical effects of complex environments in vitro and vivo during production, storage and in vivo use. And occur a variety of physical and chemical properties changes, leading to increased immunogenicity, shortened half-life, and even failure.

Therefore, how to improve the stability of antibody drugs, reduce immunogenicity, prolong the half-life, and improve they’re in vivo bioavailability are key issues that urgently need to be resolved in the application of antibody drugs.

1.Antibody molecular structure and stability research significance

Antibody refers to the immunoglobulin produced by the body that can specifically bind to the antigen. Antibodies are secreted and produced by plasma cells transformed from B lymphocytes. Each B lymphocyte line can only produce one specific antibody against a specific antigenic determinant. This kind of antibody produced from a single cell line is called monoclonal antibody (mAb).

A conventional monoclonal antibody molecule is composed of two heavy chains (HC) and two light chains (LC) connected by interchain disulfide bonds to form a “Y”-shaped structure, which can be divided into three functional components: two antigen-binding fragments (Fab) and a crystal region (Fc). The two Fabs are connected to the Fc through the hinge region, and the conformational changes are more flexible than the Fc. The Fv region of the Fab is composed of a pair of variable regions (VH and VL) from the heavy chain and the light chain. Usually, the Fv region is modified by glycosylation, which determines how the antibody interacts with other components in the adaptive immunity and humoral immune system key.

Studies have found that although certain monoclonal antibody drugs show good drug activity in in vitro experiments, they will encounter the problem of reduced in vivo activity when they enter the clinical trial stage. Therefore, the issue of pharmacodynamics must be taken into consideration in the early stage of drug development.

At present, the methods to improve the thermal stability of proteins mainly include non-covalent modification, chemical modification, addition of protein stabilizers, protein engineering. In liquid state, calcium phosphate mineralized shell was formed directly on protein surface by mineralization technology to improve protein stability.

It can be seen that, while ensuring that the affinity and expression quantity of the antibody is not greatly affected, improving its stability to the greatest extent has important practical significance for the development of antibody drugs.

Strip chart and molecular structure of IgG antibody

2.Antibody stability assessment method

The stability assessment of biotechnology products usually includes biological activity analysis, molecular structure and purity analysis (including quantitative detection of degradation products), and monitoring of related parameters (such as appearance, pH value, etc.).

Combining the above data to evaluate the thermal stability, aggregation and the magnitude of intermolecular forces for sample.

In the evaluation and analysis of thermal stability, differential scanning calorimetry (DSC) is currently the most commonly used method to measure protein thermal stability The method can not only obtain the melting temperature, but also the enthalpy, entropy and free energy related to melting.

In addition, in terms of protein solubility prediction, researchers have successively proposed cross-action chromatography (CIC), affinity capture self-interaction-nanoparticle spectroscopy (AC-SINS) or clone self-interaction-biological layer interference (CSI) -BLI) and other technologies have made certain progress.

These methods assess the likelihood of monoclonal antibody crossover or self-interaction at low protein concentrations to predict the properties of monoclonal antibodies at high concentrations.

With the application of computer-aided design in the development of biomacromolecules, a large number of modeling and simulation software can be used to predict the three-dimensional structure of antibody-antigen complex for molecules with an uncertain crystal structure. Different force fields can also be used for Moleculardynamics (MD) simulations 3 to obtain more detailed information about binding interactions, stability, and to make it easier to calculate non-covalent bond energies (hydrophobic, electrostatic, non-polar, and binding energies).

3.Stability modification plan

3.1 Modification of antibody molecular structure

Structural modification based on the chemically modified sites of antibody molecules has always been an important direction for stability optimization. Among them, the deamidation of the CDR region of an antibody may lead to the loss of the antigen binding function.

Studies have been gradually elucidating the mechanism of deamidation and chemical modification and their effects. Moreover, studies have confirmed that the deamidation rate of Gln is much slower than that of Asn. Therefore, removing the deamidation site or reducing the probability of the deamidation effect by mutating Asn to Gln is regarded as a solution4.

3.2 Production process optimization

Studies have pointed out5 that antibodies will degrade in different ways under over-acid or over-alkali conditions. In pH 6.8 immunoglobulin (IGIV) preparations for intravenous injection, this instability caused by pH can be improved by adding maltose stabilizers.

Surfactants are usually added to monoclonal antibody drug formulations to reduce the exposure of the hydrophobic region, or to reduce protein interactions and interface-induced aggregation by competing for adsorption sites. Among them, the commonly used nonionic surfactants are polysorbate 20 And polysorbate 80 7.

In addition, certain amino acids are often used as excipients to protect aggregation. The commonly used arginine (Arg) can increase the solubility of proteins and protect them from aggregation induced by light and high temperature.

It is often more economical to improve the storage or packaging of antibody drug products. So far, the method to prevent protein from interacting with the surface of the container is to coat the surface, that is, surface passivation.

Coatings can be roughly divided into two categories: single-layer coatings (more commonly used) and multi-layer coatings (less controllable). The more commonly used coating polymers include ethylene glycol or ethylene oxide11. In addition, the use of polar or neutrally charged polymer coatings will also be able to reduce protein adsorption12.


Therapeutic monoclonal antibody drugs are the research and development hotspot in the field of biomedicine at present, and on this basis, single-chain antibody (SCFV), single-domain antibody, an antibody-drug conjugate (ADC), and other drugs applied to various organ system diseases have been approved and marketed successively.

It’s a crucial problem that how to improve the stability of the drug without change drug targeting , balance effect and immunogenicity. And as far as possible extend the half-life of the drug, mantain effective blood concentration. Antibody stability is affected by many factors such as environment, formula, own structure and production operation, and effective evaluation of antibody stability is a prerequisite for individualized transformation.

Stability evaluation should not only be defined based on the presence or absence of degradation products or the concentration of stable molecules, but should include the following three aspects: the evaluation of physical stability studies should cover the number of aggregates and fragments and the structure; chemical stability studies should Pay attention to protein degradation; biological stability studies should ensure that the activity of the monoclonal antibody on the target is consistent with its physical and chemical stability.

In-depth discussion of factors affecting antibody stability and evaluation methods will help rational optimization of antibody drugs and the development of new drugs.


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[6]Hogiri T, Tamashima H,Nishizawa A , et al .Optimization of apH-shift control strategy for producing monoclonal antibodies in Chinesehamster ovary cell cultures using a pH-dependent dynamic model[J].J BiosciBioeng,2018,125(2):245-250.doi:10.1016/j.jbiosc.2017.08.015

[8]Toth RTt, Pace SE, Mills BJ , et al .Evaluation of Hydrogen ExchangeMass Spectrometry as a Stability-Indicating Method for Formulation ExcipientScreening for an IgG4 Monoclonal Antibody[J].J PharmSci,2018,107(4):1009-1019.doi:10.1016/j.xphs.2017.12.009

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