Biopharmaceuticals Flashcards
Biopharmaceutical definition
drug product manufactured from biological sources
small molecule to mAB increasing in?
increasing complexity
Biopharmaceutical characteristic: MW impurities dosing route dose toxicity specificity immunogenicity
high MW unfamiliar impurities IV dosing optimal biologic dose uncertain chronic toxicity species specific immunogenic
Recombinant DNA technology
Gene of interest extracted from HUMAN cell
Sticky ends used to insert into bacterial chromosome.
Chromosome is inserted into bacterial cell producing protein of interest
Process of manufacture
- Cell line - insertion to mammalian/bacterial cell
- Culture - transfer to large vessels
- Fermentation - transfer to bioreactors and nutrients added
- Purification - Biologic separated from biomass leading to pure solution
- Conjugation - with small molecule then further purification
- Formulation - into stable form then packed into vials
Biopharmaceutical pharmacokinetics: Tissue penetration Binding Degradation Renal clearance Unbound concentration Pharmacokinetics
Poor binding implies clearance Proteolytic degradation Renal clearance uncommon unbound concentration can exert effect and immunogenicity PK nonlinear, dependent on PD
Elimination pathways
Refer to table on sticky note
CQAs
Critical Quality Attributes
structural (chemical, physical, biological) and microbiological characteristics which relate to structural characteristics of the protein or peptide that defines the biopharmaceutical.
major biophysical structural changes:
loss of drug material from solution and
formation of soluble and insoluble (precipitates) aggregates).
Tiers of biopharmaceutical characterisation. Not representative of higher order structures etc
4- NMR
3- MS, SAXS (small angle X-ray scattering)
2- DSC, AUC
1- UV fluorescence, particle analysis
Affecting downstream processing
Resolution of properties of purification processes
detection and quantification capabilities
Regulation of biologics
EU directive
ICH Q5E
analytical testing,
biological assays, and, in some cases, nonclinical and clinical data.
stability, critical points in the manufacturing process, biological assays to evaluate the maintenance of the compound’s effects.
UV
Peptide maps
Primary through to quaternary plus their methods of analysis
Primary - peptide map
2ary - FT-IR (fourier transfer- infrared spectroscopy)
3ary - near UV CD (circular dichroism)
4ary - NMR
Steps in process - QA of final product
Impurities removal
Batch analyses,
Process validation/evaluation
Characterisation and stability
Comparable
Bio-similar definitions
comparable = highly similar quality attributes before and after manufacturing process changes bio-similar = biological medicine highly similar to another already approved medicine
What are higher order structures and their significance in biosimilar medicine
2ary to 4ary protein structures.
These must be analysed and if found to be significantly changed - the medicine is no longer a biosimilar
Reproduce antibody structure (found in folder)
…
Different classifications of antibodies (Ig?) based on their constant regions
A D E G M
mAb production
- Mouse challenged with antigen
- spleen cells produced from mouse fused with myeloma cells
- hybridomas produced and cultured in HAT medium
- positive cells selected and mAbs harvested
3 common methods of mAb isolation
EXPLANATION OF METHODS ON SLIDE 8 of lecture 11
- Phage display
- B cell immortilisation
- single-cell expression cloning
% mouse of chimeric and humanised
chimeric = 25% humanised = 10%
slide 10
mab naming
Evaluation of mAb stability (3 types)
- Chromatographic methods (.g., routine batch analyses, in-process control, process validation/evaluation data, characterisation and stability) - POLARITY/CHARGE changes
- Electrophoretic methods
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE)
Isoelectric focusing (IEF)
Capillary electrophoresis (CE)
Membrane-confined electrophoresis(MCE)
CHARGE changes - Spectroscopic methods
Ultraviolet (UV)
Circular dichroism (CD)
Fluorescence spectroscopy (FS )
Fourier-transform infrared spectroscopy (FTIR)
MOLECULES or GROUPS WITH EM RADIATION
additional stability tests for mAbs
Mass spectrometry (MS) Surface plasmon resonance (SPR) Analytical ultracentrifugation (AUC) Dynamic light scattering (DLS) Differential scanning calorimetry (DSC) Field flow fractionation (FFF)
Rituximab - accepted and hypothesised
anti-CD20 chimeric antibody
direct lysis
complement-mediated cytotoxicity (C3-C3b-MAC)
FC1R/RC-mediated opsonic phagocytosis or ADCC
Hypothesis - supress disease injury through promotion elimination of circulating and possibly lymphoid-tissue-associated B cells
Rituximab care in follicular lymphoma
- Asymptomatic - ritux. monotherapy
- Mild -Ritux. + radio-immunotherapy
- High tumour burden - immunotherapy, RCHOP, R-CVP +/- ritux.
CQAs -Analytical methods. Proof of similarity
- STRUCTURE - higher order (NMR, CD, FTIR)
- STABILITY/Post-translational modifications (expansion on this in notes - related to glycans)
- BIOLOGICAL ACTIVITY - Binding, ADCC and CDC assays
- IMPURITIES - CEX - acid/base variants
- peptide mapping deamidation
- oxidation, mutation, glycation
- SEC/FFF/AUC aggregation
For biosimilar approval (3)
- high analytical similarity - amino acid sequence, comparable glycan variants, aggregates, deamidation, stability, purity
- Biosimilarity (PK/PD/Preclinical) - therapeutic effect the same
- Extrapolation of indications - must be functionally the same for 1-2 indications and these scientifically evaluated