P1: Formulation Of Biotech Products

Front 1

Formulation Strategies for Biotech Products

Back 1

Buffers (pH & ionic strength of buffer will interact with protein structure/function)

Stabilizers

Freeze drying

Front 2

Protein Solubility/Stability
pH

Back 2

Proteins are least soluble in aqueous solutions when pH is near pI

SOLUBLE- Charged protein molecules interact with solvent

INSOLUBLE-neutral protein molecule will self associate and lead to aggregation

Front 3

Protein Solubility/Stability
aggregation

Back 3

Aggregation occurs from intermediate because protein is partially unfolded exposing hydrophobic cores that will interact with each other and aggregate

Front 4

Protein Solubility/Stability
Ionic Strength

Back 4

LOW ionic strength solubility INCREASES

HIGH ionic strength solubility DECREASES

Front 5

Preventing Physical Degradation
Stabilizing Native Conformation

Back 5

Specific Stabilizers

Sugars

Polymers

Surfactants

Front 6

Stabilizing Native Conformation
Sugars and Surfactants

Back 6

Increase CMC (Critical Miscele Concentration) to form a layer around protein (miscele) to stabilize the protein and prevent aggregation

Front 7

Sugars and Mallard Rxn

Back 7

REDUCING sugars will interact with Arg and Lys to form covalent bond which destabilize the protein and form a brownish color

Front 8

Example of Surfactant and potential incompatibilities

Back 8

Epoetin alfa & PRCA
Had a formulation change from Human Serum Albumin to PS80 to change from vial to prefilled syringe.
Leachables from rubber stopper in the presence of PS80 lead to aggregation which lead to an immunogenic response killing patient in the end
Rubber stoppers were coated to prevent leaching

Front 9

Chemical Degradation of Biotech Products

Back 9

Oxidation
Deamidation
Disulfide Exchange
Isomerization
Beta elimination
Cross linking
Fragmentation

Front 10

Oxidation

Back 10

OLA
Optimize pH
Lower temp to prevent mobility
Antioxidant (ascorbic acid)
AVOID
EDTA (will bind to metals that can catalyze a redox)
a.a.- methionine and cysteine

Front 11

Deamidation

Back 11

ODS
Optimize pH and Ionic strength
Decrease mobility (stabilize native conformation, lower temp, lyophilize)
Screen buffers

Front 12

Optimize pH

Back 12

Hydrogen peroxide may be added to a protein to induce oxidation at different pH levels and pH where oxidation is reached is used to formulate the protein

Front 13

Isomerization
Beta-elimination
Cross-linking
Fragmentation

Back 13

ODD
Optimize pH
Decrease mobility(stabilize native conformation, lower temp, lyophilize)

Front 14

Disulfide exchange

Back 14

same as Isomerization with addition of scavengers such as Glutathione that prevent proteins from binding each other

Front 15

Preservatives
(Antimicrobials)

Back 15

Benzyl alcohol
Phenol

Front 16

Antimicrobials Stability

Back 16

Antimicrobial must be high enough to remain useful but not to high as to make its way into the micele created by surfactant which will destabilize protein

Front 17

Lyophilization

Back 17

Reduces water content in protein vials and decrease mobility of protein

Front 18

Lyophilization Process

Back 18

1. Freeze
2. Reduce Pressure
3. Primary Drying
4. Secondary Drying

Front 19

Lyophilization Primary Drying

Back 19

Removes FREE water
vapor is removed through a condenser
Product must stay below Tc (COLLAPSE TEMP)

Front 20

Lyophilization Secondary Drying

Back 20

Removes BOUND water
Product must stay below Tg (GLASS TRANSITION TEMP)

Front 21

Excipients for Freeze Drying

Back 21

Buffers (avoid PO4)
Cryo and Lyo protectant (nonreducing sugars)
Bulking agent (mannitol, glycine)
Surfactant

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Cryo Protectant for Freeze Drying

Back 22

Cryo protectants protect against freezing

Front 23

Lyo Protectant for Freeze Drying

Back 23

Lyo protectants protect against drying

Front 24

Non-reducing Sugars for Freeze Drying

Back 24

WHen drying non-reducing sugars replace water to help stabilize the protein

Front 25

PO4 buffers for Freeze Drying

Back 25

Dibasic PO4 buffers will form their monobasic form which will decrease pH of solution, increase solubility, increase mobility, increase interactions with other protein molecules to form aggregates

Front 26

Bulking Agents for Freeze Drying

Back 26

Added to the formulation to give protein drug more bulk since freeze drying will decrease bulk of the drug

Front 27

Surfactants for Freeze Drying

Back 27

used not only for freeze dried products but also for reconstitution medium in protein
Keeps protein stabile during reconstitution because surface tension of water can effect conformation of protein

Front 28

Buffering Agent

Back 28

Citrate, Phosphate, Histidine

Maintain pH of the formulation

Front 29

Tonicifiers

Back 29

NaCl, KCl, glycine

Provide tonicity for solution

Front 30

Sugars

Back 30

Sucrose, Trehalose, Sorbitol, Mannitol,

Stabilize native conformation

Also used as cryo and lyo protectants and bulking agents in lyophilization

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Surfactant

Back 31

PS80, PS20, pluronic F68, pluronic F127

Bind and stabilize native conformation

Protect against stress induced by air water interface during reconstitution or shear

Protect against adsorption

Front 32

Antioxidant

Back 32

EDTA, ascorbic acid, cysteine, glutathione, methionine

Protect against oxidation

Front 33

Amino acids

Back 33

Arg, Gly, His

Stabilize native conformation and act as tonicity agents

Front 34

Bacteriostatic agent (Preservative)

Back 34

benzyl alcohol, phenol, m-cresol

inhibits bacterial growth (multi dose)

Front 35

Metals

Back 35

Calcium, copper, magnesium, zinc

Stabilize native conformation, particularly for metal binding proteins

Front 36

Polymers

Back 36

Heparin, dextran, sulfate

Stabilize native conformation like sugars

Front 37

Warning Labels

Back 37

Shear "Do not shake"
Temp "keep refrigerated"
Light sensitive "protect from light"
Freeze thaw "do not freeze"