Pharmaceutics and Pharmacokinetics of Recombinant DNA-Produced Agents

Recombinant DNA (rDNA) techniques have made it easier to produce extremely pure, therapeutically effective proteins. Pharmacists must be familiar with the chemistry of proteins in order to store, handle, dispense, reconstitute, and administer these protein medications because they have the physicochemical and pharmacological properties of proteins.

Proteins can be unstable due to chemical or physical factors. In the former situation, the protein might adhere to glass containers or flocculate, changing the dose the patient will receive. The type or stereochemistry of the amino acids, the location of disulfide bonds, the cleavage of the peptide chains themselves, and the charge distribution of the protein may all be altered by chemical events occurring on the protein in the latter scenario. Any of these may cause the protein to unfold (denaturate), lose its activity, and become inactive, making the molecule ineffective as a medication.

When a protein is being purified, the molecule may be exposed to acids or bases, which can cause chemical instability. Nevertheless, instability can also arise at the site of administration, for example, when a lyophilized protein is reconstituted. In order to foresee and address such issues, the pharmacist needs to understand a few ideas about the chemical and physical instability of proteins.

Chemical Instability of Proteins

Hydrolysis

The polymer chain may be broken by hydrolytic reactions of the peptide bonds. In the presence of diluted acids, aspartate residues hydrolyze 100 times more quickly than other amino acids. Asp-Pro linkages are typically more easily hydrolyzed than Asp-X or X-Asp bonds in peptides. This Asp feature is most likely the result of the Asp side chain carboxyl group having an autocatalytic activity. If Asn, Asp, Gln, and Glu are located close to Gly, Ser, Ala, and Pro, they hydrolyze incredibly quickly. When the side chain carboxyl groups are ionized at high pH, Asp and Glu hydrolyze most quickly, but Asn and Gln within these groupings accelerate hydrolysis faster at low pH.

Deamidation

The hydrolytic processes that Gln and Asn experience cause their side chains to deamidate. These processes change residues of neutral amino acids into charged ones. Asn is changed into Asp and Gln into Glu. Although the kind of amino acid is altered, the chain is not broken. In essence, this process is primary sequence isomerization, and it may have an impact on biological function. Under pH circumstances that are neutral or alkaline, the deamidation process of Asn residues is accelerated. The accelerant is a five-membered cyclic imide intermediate created by the nitrogen atom attacking the Asn side chain’s carbonyl carbon intramolecularly. The aspartyl peptide and an isoform are the residues that are produced when the cyclic imide hydrolyzes spontaneously.

Racemization

All amino acids, with the exception of glycine, which is achiral, are capable of undergoing base-catalyzed racemization processes. Proteins produced through racemization contain a mix of L- and D-amino acid configurations. Following the abstraction of the amino acid’s α-hydrogen to create a carbanion, the reaction takes place. The stability of the carbanion regulates the reaction’s rate, as would be expected. Asp racemizes 105 times quicker than free Asn when it uses a cyclic imide intermediate. Some amino acids in a protein, in contrast, racemize roughly two to four times more quickly than their unbound versions.

β-Elimination

In alkaline circumstances, proteins containing Cys, Ser, Thr, Phe, and Lys easily undergo -elimination, which makes it easier for an α-carbanion to develop.

Oxidation

The sulfur-containing amino acids Met and Cys as well as the aromatic amino acids His, Trp, and Tyr are all susceptible to oxidation. Both when storing proteins and during protein processing, these reactions are possible. Hydrogen peroxide or molecular oxygen can oxidize methionine (CH₃-S-R) at low pH to produce a sulfoxide (R-SO-CH₃) and a sulfone (R-SO₂-CH₃). Cysteine’s thiol group (R-SH) can be successively oxidized to produce the equivalent sulfenic acid (R-SOH), disulfide (R-S-S-R), sulfinic acid (R-SO₂H), and sulfonic acid (R-SO₃H). These reactions are influenced by a number of variables, including pH. Disulfide bonds (-S-S-) can be formed from free sulfhydryl (-SH) groups and vice versa. In the disulfide exchange phenomenon, disulfide bonds break and reform in various places, resulting in improper protein folding. Activity can be eliminated by significant alterations to the peptide’s three-dimensional structure. It is thought that a variety of oxidizing enzymes are responsible for the oxidation of the aromatic rings of the His, Trp, and Tyr residues.

Physical Instability of Proteins

Protein instability is caused by a variety of factors, not just chemical ones. A protein is a big, globular polymer with certain secondary, tertiary, and quaternary structural types. Proteins don’t have a set, rigid structure. The molecule is moving dynamically, and the structure is taking a wide-ranging sample of three-dimensional space. Non-covalent intramolecular bonds can form, break, and reform throughout this motion, but the overall shape is still centered on the energy minimum that corresponds to the molecule’s most likely and biologically active conformation. The protein’s activity can be completely eliminated by any significant change in structure. This issue is not present in small medicinal molecules. Normal globular protein folding places the hydrophobic groups on the inside and the hydrophilic groups on the exterior. The protein’s solubility in water is made easier by this configuration. Changing the hydrogen bonding, charge, and hydrophobic effects of the typical protein is possible if it unfolds. The hydrophobic groups can be moved to the protein’s outside as the globular structure is lost. The unfolded protein can then experience additional physical interactions. Denaturation describes the breakdown of a protein’s spherical structure.

The facet of protein instability that has received the most research to far is denaturation. The original molecule’s three-dimensional folding is hampered as a result, maybe even at the secondary and tertiary structural levels. In contrast to chemical composition, a protein’s physical structure changes when it denatures. As the usually spherical protein unfolds, hydrophobic residues are made visible, and the original three-dimensional structure is lost. Temperature, pH, ionic strength of the medium, the presence of organic solutes (urea, guanidine salts, acetamide, and formamide), and the presence of organic solvents like alcohols or acetone are all factors that influence the denaturation of proteins. Either reversible or irreversible denaturation can occur. Reversible denaturation will take place when the denaturant is eliminated by dialysis, allowing the denatured protein to return to its original state. Generally speaking, denatured proteins are insoluble in water, have no biological function, and are vulnerable to enzyme hydrolysis. Protein denaturation may be facilitated by the hydrophobic surface that is present at the air-water interface. In drug delivery systems and intravenous (IV) bags, interfaces like these are frequently seen.

Proteins that adhere to surfaces, such as the walls of dosage form containers, drug delivery devices, ampuls, and IV tubing, are said to have undergone surface adsorption. Proteins can stick to rubber, polyethylene, glass, plastics, and polyvinyl chloride. The term flocculation is used to describe this phenomena. Particular issues of this nature are presented by the inside surfaces of IV delivery pumps and IV delivery bags. Proteins that flocculate make accurate dosing impossible.

Protein molecules self-associate in aqueous solution to form dimers, trimers, tetramers, hexamers, and huge macromolecular aggregates, which lead to protein aggregation. In addition to solvent composition, ionic strength, and dielectric characteristics, self-association is influenced by the pH of the medium. Protein aggregation may also be brought on by moderate concentrations of denaturants (below the level required to trigger denaturation). Aggregation is a common occurrence for partially unfolded intermediates. Immunoglobulin for injection, for example, is a concentrated protein solution that can clump over time when kept on a shelf. The pharmacist’s first indication that the antibody solution is flawed is the presence of particles in the preparation.

Denaturation frequently co-occurs with precipitation. On the walls of an infusion device or its dosage form container, insulin produces a finely divided precipitate that has been the subject of extensive research. It is thought that denaturation of insulin occurs at the air-water interface, promoting precipitation. Zinc ion concentration, pH, and the presence of adjuvants, like protamine, also have an impact on how insulin precipitates.

Immunogenicity of Biotechnologically Produced Drugs

By their very nature, proteins are antigens. When given in a higher dose that would be utilized as a medicine, a human protein that is harmless at its normal physiological concentration may have entirely distinct immunogenic characteristics. A biotechnology-derived protein will differ among numerous key epitopes unless it is tailored to be 100% complementary to the human version.

The protein’s amino acid sequence might be altered (substitutions of one amino acid for another). Amino acid insertions or deletions, N-terminal methionyl groups, improper or atypical folding patterns, or oxidation of a sulfur-containing side chain of a methionine or cysteine are some examples of potential modifications. Moreover, when a protein has been created by employing a bacterial vector, a finite quantity of immunoreactive material may get into the final product. The antigenicity of a protein made through biotechnology is influenced by each of the previously mentioned factors. When given to a human patient, the protein will cause the host’s immune system to respond and neutralize it exactly like it would to a microbial attack.

For this reason, research has been done to develop medications made entirely of human protein, like insulin, which patients will need to take for a long time. Also, some of the most promising monoclonal antibodies (MAbs), which are created in mice using humanized genes to prevent a human reaction to the mouse antibody, are among the most promising biotechnology products.

Delivery and Pharmacokinetics of Biotechnology Products

The medicinal chemist and pharmacist should be worried about the ADME (absorption, distribution, metabolism, and excretion) parameters of protein medicines as they should be with any other drug class. Drugs developed through biotechnology include complexity that is not present in “conventional” low-molecular-weight medicinal compounds. To calculate the pharmacokinetic and pharmacodynamic parameters for a specific protein, ADME parameters are required. These factors are crucial for any drug’s calculation of the optimum dose for a specific reaction, for establishing a steady state and for modifying the dose to get the best possible residence duration at the receptor (pharmacodynamic parameters).

It is difficult to get medications into the human body that have molecular weights and characteristics similar to those of proteins. Unless the medication is enteric coated, the oral route cannot be utilized with a protein since the stomach’s acidity will catalyze its breakdown. Chemically labile peptide bonds can be attacked and destroyed by proteolytic enzymes, which are found in every cell in the body. Furthermore, hydrolysis and peptidase breakdown take place at the site of administration, during membrane transport across the vascular endothelium, and at reaction sites in the liver, blood, kidneys, and other bodily tissues and fluids. By co-administering peptidase inhibitors or saturating these enzymes with high medication doses, these enzymes can be avoided. Furthermore, sulfur oxidation and the oxidative metabolism of aromatic rings can take place. Once the various amino acids are absorbed into new peptides, proteins often break down into little fragments that are easily hydrolyzed.

The propensity of medications to attach to plasma proteins like serum albumin makes a pharmacokinetic profile vulnerable to major problems. In this case, they go into a fresh biodistribution compartment and may eventually slowly leave it. Nowadays, protein medicines can be administered primarily by subcutaneous (SC) and intramuscular (IM) methods.

The goal of much current research is to increase the bioavailability of peptide medications. Interleukin-2 (IL-2) conjugation with PEG is one instance of this. In comparison to IL-2 alone, these so-called pegylated proteins often exhibit slower elimination clearance and a prolonged t_1/2. Adding a substitute sugar moiety to the peptide is another tactic being used. The sugar moiety will modify the drug’s partition coefficient, most likely increasing its water solubility.

Engr. Dex Marco Tiu Guibelondo, B.Sc. Pharm, R.Ph., B.Sc. CpE

Editor-in-Chief, PharmaFEATURES