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AGGREGATION OF THERAPEUTIC PROTEINS (W.Wang, C.J.Roberts, Chapters 3 and 4) - PowerPoint PPT Presentation

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AGGREGATION OF THERAPEUTIC PROTEINS (W.Wang, C.J.Roberts, Chapters 3 and 4). AGGREGATION is a natural consequence of the response of a protein molecule to changes in itself and its environment.

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AGGREGATION is a natural consequence of the response of a protein molecule to changes in itself and its environment

Proteins have shown to contain short regions in their sequences (APRs) that are particularly prone to aggregation. The APRs contribute significantly toward the tendency of the protein to aggregate and may not be evolutionarily conserved among homologus proteins.

t°C pH ionic strength freeze/thaw agitation/shear

sequence mutations chemical degradation (deamidation, oxidation, clipping)

Computational methods to predict APRs in proteins

Dynamic energy landscapes: the energy landscapes change as the proteins respond to perturbations in their environment. Changes in the protein itself can also move its energy landscape. Result: conformational population shifts

The molecular origins of aggregation are similar between small peptides/proteins and large biotherapeutic molecules such as mAbs

Goal: Improvement of desirable mAb features

1. protein solubility -> greater expression levels in the cell lines (potency and specificity), achieving high concentration dosage forms.

2. protein native state stability via elimination/mitigation of APRs may increase shelf life of the product

External factors affecting protein aggregation(Chapter4 – W.Wang, N.Li, S.Speaker)


Solid state condition and composition




Conditions and composition of the solution (formulation buffer)


Processing steps

Solid state pH Excipients and level Physical state of the solid Moisture content

Fermentation/expression Unfolding/refolding Purification Freeze/thaw Shaking and shearing Pressurization Formulation/filling Preparation of modified protein or delivery systems

pH Buffer type and concentration Ionic strength Excipients and level Protein concentration Metal ions Denaturing and reducing agents Impurities Containers/closures Sources of proteins Sample treatment Analytical methodologies

Protein aggregation pathways

Indirect aggregation through chemical degradation (path 3)

Direct aggregation through protein self-association (path 2a) or chemical linkages (path 2b)

Indirect physical aggregation through formation of unfolding intermediates (path1)

Protein self-association

Formation of intermediates



Theoretically, the unfolded/denatured (U) state of a protein can form aggregates directly (true for many proteins that have been shown to be largely in the unfolded state naturally or to posess only two apparent states, N and U). However, most protein drugs are in folded states and aggregation contribution from the unfolded states is not significant

B22 osmotic second virial coefficient describes protein-protein interactions

Aggregation: formation of irreversible HMW species from the non-native monomer (non-native: partial or complete loss of the native structure, confers irreversibility to the aggregates formed.

Self-association: reversible formation of HMW species in which monomers in their native conformation are held together by non-covalent bonds.

Indirect physical aggregation through formation of unfolding intermediates (path1)

Do not aggregate easily because the hydrophobic side chains are either buried out of contact with water or randomly scattered.

Under normal conditions:

N state (native, folded)

I state (unfolding intermediates)

D state (completely unfolded/denatured)

Precursors of aggregation process because they expose more hydrophobic patches and have a high flexibility relative to the folded state

Aggregates: all non-native protein oligomers, whose sizes are at least twice as that of the native protein.

The initial aggregates (A state) are soluble oligomers but gradually become insoluble as they exceed certain size and solubility limits (P state)

Direct aggregation through protein self-association (path 2a)

Direct physical association into reversible oligomers/aggregates from the native/folded state. Can be considered the precursors of irreversible aggregates.

Electrostatic and/or hydrophobic interactions depending on the experimental conditions. Van der Waals interactions may be present.



B22(osmotic second virial coefficient)measures theprotein’s tendency to self-associate

Protein – protein interactions (PPI) are favored over the protein-solvent interactions (measured by A2)


Non ionic species (excipients/additives as sucrose) can modify the B22 value


Ionic strength


Aggregates: dimers, trimers... which maintain the native-like state

Both pH and ionic strength affect the charge density/distribution of proteins.

Minimization of protein surface charge will likely lead to increased aggregation, regardless of the specific AA sequence

Direct aggregation through protein chemical linkages (path 2b)

  • Many chemical reactions directly cross-link protein chains, leading to aggregation. The most common: intermolecular disulfide bond formation/exchange.

  • Surface located Cys are more involved in the participation in disulfide bond formation/exchange

  • Disulfide bonded proteins with no free Cys can still undergo aggregation through disulfide exchanges via β-elimination

Reversibility of protein aggregation

The ability of the protein aggregates to dissociate (disaggregate) in an equillibrium upon reversal of the solution condition when aggregation is induced: pH, t°C, concentration of excipients (e.g.salts)

  • Reversible: early aggregation

  • Irreversible: late-stage aggregation/precipitation

Protein gelation is another form of aggregation and can often occur when the solution condition favors weak interactions among protein molecules (e.g. when the solution pH is close to the protein pI)

Effects of solution conditions and composition on protein aggregation

Solution pH

Buffer type and concentration

Ionic strength


Protein concentration

The solution conditions/factors can potentially influence protein aggregation directly or could indirectly contribute to the overall rate of protein aggregation in solution

Solution pH

(+) charge

pI (no net charge)

(-) charge

the pH at which solubility is often minimal

Dispersive forces that may lead to aggregation/precipitation

Repulsive electrostatic interactions between (+ +) or (- -)

Indirect effect: interactions with aggregationexcipients/additives

Effect on the aggregates morphology (how close it is to the protein pH)

Effect on the aggregation pathway (alters charge – charge interactions, partial/complete unfolding, chemical degradation rates and pathways)

Solution pH


Impact of freezing on pH of buffered solutions and consequences for monoclonal antibody aggregation - Parag Kolhe, Elizabeth Amend, Satish K. Singh (Article first published online: 28 DEC 2009, DOI: 10.1002/btpr.37)

Each 0.4 mL of HUMIRA contains 20 mg adalimumab, 2.47 mg sodium chloride, 0.34 mg monobasic sodium phosphate dihydrate, 0.61 mg dibasic sodium phosphate dihydrate, 0.12 mg sodium citrate, 0.52 mg citric acid monohydrate, 4.8 mg mannitol, 0.4 mg polysorbate 80, and Water for Injection, USP. Sodium hydroxide added as necessary to adjust pH. il pH di cui parlano è 5