Formulation of biopharmaceuticals Flashcards
examples of biopharmaceuticals
- Monoclonal antibodies
- ADC (antibody-drug conjugate)
- Interleukins
- peptides
- virus-like particles
Name the 3 broad groups of protein therapeutic biopharmaceuticals
- protein therapeutics with enzymatic or regulatory activity
- protein therapeutics with special targeting
- protein vaccines
examples of how protein therapeutics with enzymatic or regulatory activity are used
- replacing a protein that is deficient or abnormal
- augmenting an existing pathway
- providing a novel function or activity
examples of how protein therapeutics with special targeting are used
- interfering with a molecular pathway or an organism’s physiology
- delivering other compounds or proteins
examples how protein vaccines are used
- protecting against a deleterious foreign agent
- treating an immune disease
- treating cancer
How are mABs classified?
4 parts of name
1) unique prefix
2) prefix letters related to type of target (part of body/tissue)
3) prefix reflects source of the variable chain (e.g. mouse, rabbit)
4) suffix -mab
How are biologics formulated?
How are they administered?
Issues?
- either as a liquid formulation or lipophilised (to be reconstituted before use)
- administration is either subcutaneous or IV
- issues often linked to frequency of administration
- adverse effects
Formulation of proteins and mABs
Solid form advantages/disadvantages
- dose and injection volume adjustable
- can be developed as multi-use formulations
- can be more expensive to couple a solid form to a delivery device (e.g. dual chambers)
Formulation of proteins and mABs
Liquid form advantages/disadvantages
- more convenient to end user
- better patient compliance
- better accuracy
- chemical degradation hydrolysis therefore less stable, limit shelf life, manipulation etc.
- physical stability more difficult to control: aggregation (e.g. exposure to final fill finish operations)
Approaches to formulation development of proteins and mABs
- development of stability-indicating assays
- in silico assessment of protein degradation routes
- complexity of stability determinations during formulation development (real time vs accelerated stability)
- liquid formulation development
List excipients for proteins and mABs
- buffers
- salt and tonicity modifiers
- surface active agents
- anti-oxidants
- protein stabilisers
- lypophilation development
- caveats to use of sugars as lypoprotectants
Buffers
examples and info
-acetate
-citrate
-succinate
-histidine
-phosphate
Formultions pH range 5-6.5 (IgE pI approx 8)
Buffer concentration kept low to adapt to physiological pH upon administration
Salt and tonicity modifiers
Colloidal stability
IV injection requires isotonic preparation
IM or SC injections may be able to handle hypertonic or hypotonic conditions
common excipient is NaCl
Surface active agents
- mABs flexible molecules with hydrophobic and hydrophilic regions
- unfolding leads to aggregation
- surfactants cover interfaces (air/liquid and solid/liquid) thus limit unfolding
- Polysorbates 80 and 20 most common
- PS degration may contribute to aggregation
Antioxidants
- oxidation reaction catalysed by metals
- use of EDTA to chelate metals contributes to control oxidation
- reducing agents such as glutathione can reverse oxidation
Protein stabilisers
- stabilisers are preferentially excluded from the protein’s surface leading to preferential hydration of protein
- sugars e.g. sucrose
- amino acids such as Arginine
Lypophilisation development
- Use of PEG
- Same mechanism as stabilisers: exclusion by steric hinderance and maintained upon freezing
Caveats to use sugars as lypoprotectants
- Disaccharides susceptible to hydrolysis at low pH
- hydrolysis of sucrose to glucose and fructose at pH 5
List stability issues with mABs and proteins
- chemical degradation
- physical degradation
- chemical oxidation
- physical stability
Chemical degradation
- most common routes are oxidation, de-amidation, Asp isomerisation and cross-linking
- alteration of residue may lead to conformational changes and/or binding (ex. deamidation of Xolair)
- Deamidation is a common route and is dependent on amino acids that flank the amide residue
- Asp isomerisation may lead to degradation in one of the CDR (Herceptin 90% loss of activity)
Chemical oxidation
- oxidation is most common route
- Amino acid susceptibles are: Met, Tyr, His, Trp and Cys
- Met oxidation in mABs frequent
- His oxidation via oxidation and metal-catalysed reactions
- Trp oxidation occurs via metal-catalysed reactions (e.g. Trp in CDR of palivisumab when exposed to UV light)
- Cys intermolecular disulphide linking occurs in several mABs
-Non-enzymatic protein degradation: fragmentation reported in mABs following storage at 37°C for 3 weeks in acidic or basic conditions
Physical degradation
- conformational changes, aggregation and surface adsorption
- conformational changes=denaturisation=unfolding
- all information to maintain protein conformation is contained in the amino acid sequence (primary structure)
sources of conformational changes
- temperature changes
- ice formation due to freeze thaw
- shear forces
- changes in ions in solution
- changes in protein-protein interactions
Physical stability - aggregation
-may originate from conformational changes induced by covalent changes (chemical) but very often related by the hydrophobic/hydrophilic issues
“Soluble aggregates”
- no particle visible in solution and aggregates cannot be removed easily by filtration
- Quantification of sub-visible aggregates (100nm-10um) may be difficult and may be immunogenic (not established unequivocally)
reversible aggregates
- self association
- may be the consequence of formulation or delivery
Why are aggregates unfavourable?
What is the WHO limit on them?
They alter pharmacokinetics and reduce the activity
WHO limits their presence to less than 5%
Protein aggregation
- protein aggregation is a consequence of protein-protein interactions
- aggregation may happen at each steps of the bioprocess, formulation, storage
- IgG2 is more prone to aggregation than other IgGs
- aggregation prediction uses accelerated stability testing
- air water interface: mABs are amphipathic molecules and hence tend to move at the interface
- Adsorption to surfaces (all solid surfaces of the container closure system) - use surface active agent to limit adsorption
Opportunities for aggregation
- cell culture
- shipping
- freeze drying/spray drying
- filtration and filling
- recovery and purification
- tangenital flow filtration
- bulk storage and freeze/thaw
Challenges for IV formulations
- leachables and extractables
- head space
- preparation bags for infusion
What are leachables?
- compounds released from a container closure system when in contact with solvent (depends on pH, temperature, salt concentration)
- includes metals, organics, volatile compounds
What are extractables?
- subset of compounds eluting from normal storage use conditions e.g. Infusion bag, tubing
- IV bags produced with different polymers (PO may be better than PVC)
- bags volume may also be considered
Clinical use: head space
-the pharmacists has the responsibility to ensure product stability in the final administered form and the setting of “beyond-use date” based on United States Pharmacopoeia 797
What is the beyond-use date?
-the “beyond-use” date is defined as the time the compounded sterile must be used to avoid lack of potency, contamination and safety risks
Preparation in bags for IV infusion
Dilution of the protective surfactant can lead to noticeable degradation of the product and the generation of an air-water interface on agitation was responsible for aggregate formation
Challenges for SC formulations
- small volume (1.5mlL) but high concentrations (50, 100 are more mg/ml)
- increased protein-protein interactions and risks of aggregation
- impact on flow, viscosity, internal diameter of needle is small (0.2mm)
- Glide force max (30N) relation to viscosity
- Bioavailability of SC delivered mABs may vary widely
- Development of analytical tools to characterise these solutions
use of biopharmaceuticals
- last line antimicrobials
- treat long term diseases
