Module 9 2024

03/09/2024

Purity - Charge Heterogeneity Example Method: cIEF/IEX Acidic Basic

Deamidate d

Major Chemical Degradation Pathways

Effect

Species Formed

Sialylation

COOH addition

Acidic

Deamidation

COOH formation Acidic

C-terminal lysine clipping

Loss of NH2

Acidic

Adduct formation

COOH formation or loss of NH2

Acidc

Succinimide formation

Loss of COOH

Basic

Methionine, cysteine, lysine, histidine, tryptophan oxidation

Conformational change Conformational change

Basic

cIEF = capillary electrophoresis IEX – ion exchange chromatography

Disulphide-mediated

Basic

Asialylation (terminal galactose)

Loss of COOH

Basic

Khawli et al, Mabs 2;6 1-12 2010

C-terminal lysine and glycine amidation

NH2 formation or loss of COOH

Basic

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Purity – Aggregates (HMWs) Example Method: SE-HPLC

- Aggregates are defined as immunity- provoking factors in the FDA Guidance ‘Immunogenicity Assessment for Therapeutic Protein Products’ (Aug 2014).

- The WHO recommends ‘Generally, not less than 95 % of the immunoglobin present should be in form of molecular monomers and dimers.

- Typically for a mAb aggregates are quantitated by Size exclusion (SEC) HPLC method

- In Europe an orthogonal aggregate determining method such as light scatter or Analytical ultracentrifugation should be included (AUC).

Monomer

HMWS Aggregat e

LMWS Fragment

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