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Pharmaceutical Dosage Forms and Drug Delivery
frequency of administration, patient compliance, and the physicochemical properties of the drug.
For example:
• The IV route is preferred for rapid onset of administration, whereas the SC route can be used for
sustained drug delivery devices. Thus, sustained drug delivery devices such as poly(lactide- coglycolide) (PLGA)- entrapped drugs are often designed for SC administration.
•
•
•
-
aration of concentrated protein solutions can lead to high viscosity— which could make deaeration upon
25.6.2 Type of Formulation
Selection of the type of protein formulation depends on several factors, such as follows:
• Disease condition: For example, the requirement of patient self- administration (SC route pre-
ferred) versus administration by a health care professional in a hospital setting (IV route preferred)
• Drug half- life: Rapidly cleared drugs must be administered as an IV infusion to obtain sustained
plasma concentrations.
• Patient population: The Age of the patient may determine the kind of delivery devices that may be
• Route of delivery, such as IM, IV, SC, intraperitoneal, topical, inhalation, or nasal. IV formulations can
further be IV bolus or IV infusion. Inhalation formulations can be dry powder-based or solution-based.
• Drug dose, solubility, stability, and other physicochemical properties.
Proteins and peptides for parenteral administration are typically formulated as ready- to- use aqueous
isotonic dextrose solution or isotonic sodium chloride solution immediately before administration.
Proteins and peptides for inhalation and nasal routes of administration are typically formulated as dry
powders. The details of dry powder formulations will not be discussed in this chapter.
25.6.3 Formulation Components
Developing a suitable pharmaceutical formulation of a protein usually involves screening a number
of physiologically acceptable buffers, salts, chelators, antioxidants, surfactants, cosolvents, and
preservatives (Table 25.3). Formulation components are selected to address one or more requirements for
protein formulations, such as follows:
•
•
• Physical stability improvement by the addition of polyhydric alcohols, carbohydrates, and amino
acids. Adding these components to aqueous solutions of proteins leads to their hydrogen bonding
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Protein and Peptide Drug Delivery
TABLE 25.3
447
Typical Excipients in Protein Formulations
Category Type Functionality Examples
Buffering agents Nonamino acid
buffers
Amino acids glycine, histidine
Tonic agents Salts
Hydrophilic
additives
Surfactants Reduce surface tension,
Preservatives Antioxidants
Sugars
Other polyols Glycerol, cyclodextrins
Amino acids
Hydrophilic
polymers
Cosolvents Increase protein solubility Ethanol
Antimicrobial
preservation
Ensure optimal pH control Acetate, citrate, carbonate, HEPES, maleate,
phosphate, succinate, tartrate, TRIS
Sodium chloride, potassium chloride, calcium
isotonicity
formulations, and isotonicity
interactions
Polymer matrix in solution Dextran, PEG
protein substrate
Heavy metal binding EDTA, DTPA, EGTA
Antimicrobial agents
chloride, magnesium chloride, sodium
gluconate, sodium sulfate, ammonium
Glucose, fructose, lactose, maltose, mannitol,
sorbitol, sucrose, trehalose, inositol
Alanine, arginine, aspartic acid, lysine,
proline
Polysorbate, poloxamer, sodium lauryl
sulfate
Ascorbic acid, citric acid, glutathione,
m- cresol, methylparaben, propylparaben
Abbreviations:
tetraacetic acid.
•
the protein surface area in contact with the solvent.
• Electrostatic interactions in proteins may be modulated by altering the solvent polarity and dielectric constant to change protein electrostatic interactions in solution, which may reduce the association tendency of a protein.
• Antimicrobial agents may be added to large- volume parenteral (LVP) solutions or multidose
vials where repeated puncturing for dose withdrawal is expected to preserve aqueous solutions of
proteins against bacterial and fungal growth.
• Chelating agents and antioxidants may be added to prevent metal and/ or oxidation- induced chemical instability.
• Osmolarity control is required for parenteral formulations. This is often achieved by using salts,
buffers, and sugars.
25.6.4 Manufacturing Processes
25.6.4.1 Protein Solution
A typical manufacturing process of protein solution involves
1.
2. Formulation (dilution and addition of excipients).
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Pharmaceutical Dosage Forms and Drug Delivery
3.
4. Filling of drug products in vials or syringes.
5.
Labeling and packaging.
7. Storage and shipment of drug products.
8.
Many of these processes may affect formulation stability. For example:
• Exposure to light and shear during inspection and transportation can lead to the formation of
microbubbles in the formulation, which can increase the propensity for aggregation and oxidation.
• Protein may interact with the silicone oil typically used in syringes for smooth barrel movement,
•
the leaching of metal ions from manufacturing vessels into the protein formulation can lead to protein
25.6.4.2 Lyophilization
Many proteins are unstable in solution and may not yield an acceptable shelf life, even under refrigerated
products. For example, high concentrations of reacting species in the protein microenvironment can be
required to ensure cake integrity and rapid reconstitution.
The role of residual moisture
The amount of moisture adsorbed on each protein as a monolayer can be determined by the Brunauer–
protein to shield their highly polar groups, which would otherwise be exposed, leading to aggregation and
opalescence upon reconstitution. On the other hand, high moisture content could increase plasticity in the
system, leading to high reactivity and compromising the physicochemical stability. For example, insulin,
tetanus toxoid, somatotrophin, and human albumin aggregate in the presence of moisture, which can lead
to reduced activity, stability, and diffusion.
protecting against dehydration. Polyvinylpyrrolidone (PVP) and bovine serum albumin (BSA) protect
the reorientation of polymeric proteins and other components with excipients and provides better cake
condensate weight or volume at the vacuum pump (which indicates the amount of water removed), and/
or change in product temperature through temperature probes inserted in vials (which indicates changes
in the heat of sublimation).
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Protein and Peptide Drug Delivery
Review Questions
449
25.1 Most protein drugs have poor oral absorption because of their
A
B Poor transport via the paracellular route
C Negligible passive diffusion
D Degradation in the GI tract
E All of the above
25.2
A
B
C Addition of certain polymers
D Addition of sugars to the formulation
E All of the above
25.3
A Proteases
B
C Oxidases
D Phosphorylases
25.4
A EDTA
B EGTA
C DTPA
D Ascorbic acid
E Citric acid
25.5 Which of the following levels of protein structure is only possible for proteins that have more
A Primary structure
B Secondary structure
C Tertiary structure
D
E All of the above
A Antihuman CD31 mouse monoclonal antibody
B
C Antihuman CD31 human monoclonal antibody
D Antihuman CD31 mouse domain antibody
E Antihuman CD31 mouse- human chimeric monoclonal antibody
25.7 Aqueous protein solubility is least likely to depend on
A pH
B Salt concentration
C Isoelectric point
D Cosolvent content
E Preservative content
25.8
A Improving chemical stability of the protein
B Ease of handling and transportation
C Economically cheaper option of protein formulation
D Reducing the kinetics of degradation reactions
25.9
A Asparagine
B Cysteine
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Pharmaceutical Dosage Forms and Drug Delivery
C Glutamine
D Histidine
E Proline
25.10
that apply.
A Asparagine
B Cysteine
C Glutamine
D Histidine
E Proline
25.11
A Asparagine
B Cysteine
C Glutamine
D Histidine
E Proline
25.12
A The secondary structure of proteins refers to the conformation of the polypeptide backbone.
B Oxidation of methionine to methionine sulfoxide can be reversed with a suitable redu-
cing agent.
C The peptide bond between aspartic acid and proline are susceptible to hydrolysis at
acidic pH.
D The amide groups of asparaginyl and glutaminyl residues are labile at acidic pH.
E
F Exposure to hydrophobic surfaces may promote protein aggregation.
25.13 Protein denaturation
A Can be either reversible or irreversible
B Can be caused by exposure to hydrophobic surfaces
C Can be induced by extreme pH
D All of the above
25.14
25.15
25.17
25.18
25.19
FURTHER READINGS
Pharmaceutical
Formulation Development of Peptides and Proteins, London: Taylor & Francis Group, pp. 89– 112.
Physicochemical Principles of Pharmacy, 4th ed., London: Pharmaceutical
Press.
-
Pharmaceutical Formulation Development of Peptides and
Proteins, London: Taylor & Francis Group, pp. 70– 88.
Theory and Practice of Contemporary Pharmaceutics, Boca Raton,
FL: CRC Press, pp. 525– 547.
Lee V.H.L. (Ed.) (1991) Peptide and Protein Drug Delivery
Walsh G. (2002) Proteins: Biochemistry and Biotechnology
Pharmaceutical Biotechnology, 2nd ed., London: Taylor
& Francis Group, pp. 5– 29.
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26
Antibody Therapeutics
On completion of this chapter, the students should be able to
1. Describe different types of antibodies.
2. List types of antibody therapeutics.
3.
4.
formulations.
26.1 Introduction
antigens
of an invading pathogen, thus marking it for phagocytosis) or complement- mediated destruction. An anti-
immunological, physiochemical, and structural characteristics: IgM, IgG, IgA, IgD, and IgE. IgGs, com-
the immunological mechanism of action. Antibodies inhibit extracellular pathogens by obstructing their
“docking receptors,” which are used to adhere to host cells, thus preventing their entrance and infection.
and stably, presenting a more challenging defense against the pathogen. Avidity refers to the strength
but also depends on the number of identical binding sites and other factors. Multimeric antibodies, like
taneously. Antibodies bind to epitopes corresponding to a small region (the surface area of about four to
six amino acids) on a macromolecule. It is possible that many macromolecules have the same epitope,
allowing antibodies to attach to different macromolecules, a phenomenon known as cross- reactivity.
Advancements in understanding antibody structure led to the development of single- chain antibodies
LEARNING OBJECTIVES
DOI: 10.1201/9781003389378-30
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Pharmaceutical Dosage Forms and Drug Delivery
attached payload. This chapter outlines numerous antibody- based treatments and approaches to increase
in antibody therapeutics.
26.2 Types of Antibody Therapeutics
26.2.1 Full- Length Antibody
Antibodies are also known as immunoglobulins (abbreviated Ig) because they are immune- response
proteins that are globular proteins (compact with higher orders of structure and hydrophilic surface making
them soluble, as against brous proteins, which have predominantly secondary structure and are insol-
(
bridges. Antibody fragments consist of a constant region (designated, Fc) and a variable, antigen- binding
polyclonal,
monoclonal. Identical immune cells make mono-
clonal antibodies (mAb), whereas polyclonal antibodies are produced by a mass of immune cells that may
produce antibodies against different regions of the antigen. In industrial applications, mAb is prepared
by recombinant DNA technology in cell cultures. For human clinical applications, mAb is generally
A number of immunoglobulin (Ig) G products have been developed for therapeutic use in various
domain antibodies. Also, antibodies that can bind two different antigens are called BsABs.
The usefulness of antibodies was limited by the immune response generated by the host to the
animal species, such as mice. The antibodies generated in mice were named with the sufx ~momab.
FIGURE 26.1 Typical structure of an antibody.
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Antibody Therapeutics
TABLE 26.1
453
Types of Antibodies
Proportion of
Antibody
IgA Nose, breathing passages, digestive
IgG
IgM Blood and lymph First type of antibody made in
IgE Small amounts Lungs, skin, mucous membranes React to pollen, fungal spores, and
IgD Small amounts Tissue that lines belly or chest Not clear
Total Antibodies Where Found in the Body Function Size
Protection on the mucosal surfaces
tract, ears, eyes, saliva, vagina,
tears, and blood
most common
of the body exposed to the outside
environment
Fighting bacterial and viral infections.
Only type of antibody that can
cross placenta
response to infection. Stimulate
other immune cells
animal dander. May be involved in
allergic reactions
Smallest
Largest
helped to overcome these limitations.
• Chimeric and humanized antibodies are produced from nonhuman species whose protein sequences
• Chimeric antibodies consist of murine variable regions fused with human constant regions, resulting
These antibodies are named with the sufx ~ximab. For example, rituximab is a chimeric antibody.
• Humanized antibodies are made by grafting the murine variable amino acid domains (which deter-
These antibodies are
named with the sufx ~zumab®
advanced colorectal cancer in combination with other drugs.
• Human monoclonal antibodies can be produced using phage display or transgenic mice. Transferring
the human Ig genes into the mouse genome can produce these antibodies. These antibodies are
named with the sufx ~mumab. For example, ipilimumab is a human mAb that inhibits the checkpoint receptor cytotoxic T lymphocyte- associated antigen 4 (CTLA4) and is recommended for
advanced- stage melanoma.
Most therapeutic antibodies exert their therapeutic effects by binding to selected cellular targets, which
are then destroyed by physiological mechanisms activated by the effector functions of the antibody. In
addition, antibodies can also be used for drug delivery and targeting vehicles. Active research and devel-
antibody. For example, Adcetris®® are ADCs for tumor treatment.
26.2.2 Single- Chain Antibody
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Pharmaceutical Dosage Forms and Drug Delivery
humans. In addition, it should be noted that small animals such as mice may not consistently exhibit
assays. Further, even with the use of hybridoma, mAb production is very laborious and time consuming.
Antibodies production by recombinant DNA technology faces problems like improper folding and aggregation in the bacterial cytoplasm.
more straightforward and practical application of recombinant technology. Fab fragment is a truncated
antibody that contains all the domains: VL, VH, CL, and CH1. Fab fragments can be acquired by recom-
bridge thiols are called Fab’ fragments, whereas those lacking the thiol functional group are termed Fab
Fab fragments.
H and VL
fragment. The two domains in Fv are held together by relatively weak non- covalent interactions and,
therefore, are highly unstable. As a result, additional alterations were made to Fv fragments, including
permutated Fv (pFv) fragments. Among these, scFv fragments are superior biosensing elements. One
advantage of these is that they could retain the intact antigen binding site (paratope) while reducing
including better tumor penetration, more rapid blood clearance, lower retention times in nontarget tissue,
for diagnostic and therapeutic purposes as well as for structural studies.
The Fv fragment is the smallest unit of the immunoglobulin molecule and functions in antigen- binding
activities. An antibody in scFv (single chain fragment variable) format consists of variable regions of
heavy (VH) and light (VL
peptide linker should span 3.5 nm (35 Å) between the carboxy terminus of the variable domain and the
amino terminus of the other domain to maintain antigen- binding site integrity. In addition to linker length,
its amino acid sequence must be hydrophilic to avoid intercalation across variable domains during protein
residues like Glu and Lys for hydrophilicity. scFv antibodies could be constructed using both hybridoma
and phage recombinant techniques. However, the phage- display method of scFv production provides
mutagenesis. A wide variety of scFvs targeting proteins, carbohydrates, haptens, tumor antigens, and
viruses have been developed.
26.2.3 Bispecific Antibodies (BsAbs)
BsAbs are synthetic proteins formed through an integration of antigen recognition sites from multiple
antibodies. Targeting two antigens or epitopes can cause multiple physiological responses, which may
be independent or connected. These may function as a combination of two mABs, but drug developers
only need to produce a single molecule, and patients may require only a single therapy. Furthermore,
their cooperative or synergistic characteristics may generate more substantial therapeutic outcomes.
-
dence of resistance against infections and greater cytotoxic effects against tumors. The FDA approved the
BsAbs are developed using strategies to match heavy and light chains to avoid side products. They are
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Antibody Therapeutics
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divided into IgG- like and non- IgG- like types. IgG- like BsAbs improve solubility, stability, and biological
activity, while non- IgG- like BsAbs have therapeutic effects and low immunogenicity.
26.2.3.1 Mechanisms of Action of BsAbs
Due to their dual targeting, BsAbs may act like a binder between two cells, such as attaching immune
cells to tumor cells and allowing them to destroy them. BsAbs may also disrupt immune cell and tumor
release syndrome (CRS) by targeting the tumor microenvironment. Some BsAbs are being developed
for hemophilia A, neovascular age- related macular degeneration, diabetes, bacterial pneumonia, and
26.2.3.2 Approved BsAbs
Catumaxomab treats malignant ascites from solid tumors by targeting epithelial cell adhesion molecule
(EpCAM) and CD3. Amgen’s blinatumomab targets relapsed precursor B- cell ALL with CD3/ CD19 dual
26.3 Strategies to Overcome the Limitations of Protein Therapeutics
26.3.1 Chemically Modified
•
• Increase therapeutic ability, for example, radiolabeled antibodies and ADCs (Table 25.1).
• Increase plasma half- life, for example, by PEGylation of antibodies.
This section talks about a few distinct methods that may be used to modify proteins and peptides
chemically.
26.3.1.1 Termini Protection
Proteolysis may occur at both the N- terminal and the C- terminal of a peptide, and it can be caused by a
variety of proteases, including carboxypeptidases, serum aminopeptidases, and other proteases. Different
amino acid residues at the N- or C- terminus cause varying degrees of degradation and proteolysis.
Peptides that contain a high concentration of N- terminal Serine, Threonine, Glycine, Alanine, Valine, and
Methionine residues exhibit notable resistance to degradation in plasma. Peptide in- vivo stability can also
be increased through the addition of C- terminal amidation or N- terminal acetylation.
26.3.1.2 Backbone Modification
biological membrane permeability. It can be carried out by substitution reactions such as by exchanging
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