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

sequence mutations chemical degradation

(deamidation, oxidation, clipping)

t°C pH

ionic strength freeze/thaw

agitation/shear

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.

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

2

Conditions and composition of the

solution (formulation buffer)

1

Temperature 3

Processing steps

4

Solid state condition and composition

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

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

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 pathwaysProtein aggregation pathways

Indirect physical aggregationIndirect physical aggregation through formation of through formation of unfolding unfolding

intermediates intermediates (path1)(path1)

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

Indirect aggregation through chemical degradation (path 3)

COLLOIDAL STABILITYCOLLOIDAL STABILITYCONFORMATIONAL STABILITYCONFORMATIONAL STABILITY

Formation of intermediatesProtein self-association

B22 osmotic second virial coefficient describes protein-protein interactions

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

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 aggregationIndirect physical aggregation through formation of through formation of unfolding intermediates unfolding intermediates (path1)(path1)

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

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

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

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

Direct aggregationDirect aggregation through protein through protein self-association self-association (path 2a)(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 the protein’s tendency to self-associate

>0 (+) REPULSION

<0 (-) ATTRACTION Protein – protein interactions (PPI) are favored over the protein-solvent interactions (measured by A2)

-> AGGREGATION

pH Ionic strengthB22

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

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

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

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

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

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

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

Direct aggregationDirect aggregation through protein through protein chemical linkages chemical linkages (path 2b)(path 2b)

Reversibility of protein aggregationReversibility 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 aggregationEffects of solution conditions and composition on protein aggregation

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

Buffer type and concentration

Ionic strength

Excipients/additives

Protein concentration

Solution pHSolution pH

pI (no net charge)

(+) charge (-) charge

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

the pH at which solubility is often minimal

Dispersive forces that may lead to aggregation/precipitation

Solution pHSolution pH

Indirect effect: interactions with excipients/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)

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)

t°C

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