Pharmaceutical Biotechnology PHT 426 “Formulation of Biotech Products (1)” Dr. Mohammad Alsenaidy Department of Pharmaceutics College of Pharmacy King Saud University Office: AA 101 msenaidy@ksu.edu.sa

Introduction to Proteins Purification of Proteins Characterization of Proteins Protein’s Instabilities Formulation Examples of Protein therapeutics

Why do we formulate drugs? The overall goal of protein formulation development is to transform a highly-purified, recombinant protein solution (drug substance) into a stable, efficacious biopharmaceutical drug or vaccine (dosage form) for administration to patients. The first step is often called preformulation characterization which involves determining the protein's physicochemical properties and pathways of instability, allowing for the design of formulations containing various excipients to ensure protein stability under defined storage conditions. Specific formulations at different protein concentrations with targeted levels of various pharmaceutical excipients are then experimentally tested to ensure stability, solubility and tonicity over the shelf life. In addition, the primary container is selected (e.g., vial, cartridge or prefilled syringe) to store the protein–excipient mixture and facilitate parenteral administration by the patient or a medical professional.

Major Themes in this section Contamination. Excipients used. Shelf-life of protein based drugs. Delivery of proteins.

Contamination For pharmaceutical applications, product purity mostly is ≥99 % when used as a parenteral. Purification processes should yield potent proteins with well-defined characteristics for human use from which “all” contaminants have been removed to a major extent. Contaminants can be host-related, process-related and product-related. Special attention is paid to the detection and elimination of contamination by viruses, bacteria, cellular DNA, and undesired proteins.

Sterility Most proteins are administered parenterally and have to be sterile. In general, proteins are sensitive to heat and other regularly used sterilization treatments; they cannot withstand autoclaving, gas sterilization, or sterilization by ionizing radiation. Consequently, sterilization of the end product is not possible. Therefore, protein pharmaceuticals have to be assembled under aseptic conditions. Equipment and excipients are treated separately and autoclaved or sterilized by dry heat (>160 °C), chemical treatment, or gamma radiation. Assembly of the product is done in class 100 (maximum 100 particles > 0.5 μm per cubic foot) rooms with laminar airflow that is filtered through HEPA (high efficiency particulate air) filters. Last but not least, the “human factor” is a major source of contamination. Well-trained operators wearing protective cloths (face masks, hats, gowns, gloves, or head-to-toe overall garments) should operate the facility. Regular exchange of filters, regular validation of HEPA equipment, and thorough cleaning of the room plus equipment are critical factors for success.

Viral contamination Endogenous and adventitious viruses, which require the presence of living cells to propagate, are potential contaminants of animal cell cultures and, therefore, of the final drug product. If present, their concentration in the purified product will be very low and it will be difficult to detect them. Viruses can be introduced by nutrients, by an infected production cell line, or they are introduced (by human handling) during the production process. Appropriate screening of cell banks and growth medium constituents for viruses and other adventitious agents should be strictly regulated and supervised.

Viral contamination Methods to inactivate and remove possible viral contaminants are mandatory for licensing of therapeutics derived from mammalian cells or transgenic animals. Removal of viruses by nanofiltration is an elegant and effective technique and the validation aspects of this technology are well described. Filtration through 15 nm membranes can remove even the smallest non-enveloped viruses like bovine parvovirus. Another common, although less robust, method to remove viruses in antibody processes is by ion-exchange chromatography.

Bacterial contamination Usually the size of bacteria allows simple filtration over 0.2 μm (or smaller) filters for adequate removal. Raw materials used have to be sterilized and the products are manufactured under strict aseptic conditions. Antibiotic agents can be added to the culture media in some cases but have to be removed further downstream in the purification process. However, the use of betalactam antibiotics such as penicillin is strictly prohibited due to oversensitivity of some individuals to these compounds.

Pyogen contamination and removal Exogenous pyrogens can be derived from bacterial, viral, or fungal sources. Bacterial pyrogens are mainly endotoxins shed from gram-negative bacteria. They are lipopolysaccharides. lipid-A moiety is conserved in thousands of different endotoxins. Endotoxins are high negatively charged.

Pyogen contamination and removal They are stable under standard autoclaving conditions but break down when heated in the dry state. For this reason equipment and container are treated at temperatures above 160 °C for prolonged periods (e.g., 30 min dry heat at 250 °C). Simple 0.2 μm filtration does not remove pyrogens. Purification schemes usually contain at least one step of ion-exchange chromatography (anionic-exchange material) to remove the negatively charged endotoxins. Materials used in process such as glassware are typically subjected to a depyrogenation step prior to use often by the use of elevated temperatures (≥180 °C) in a dry heat oven.

Protein Contaminants and Product Variants Cellular DNA The application of continuous mammalian cell lines for the production of recombinant proteins might result in the presence of oncogene-bearing DNA fragments in the final protein product. If the presence of nucleic acids persists in a final preparation, then additional steps must be introduced in the purification process. Protein Contaminants and Product Variants Minor amounts of host-, process-, and product-related proteins will likely appear in biotech products. These types of contaminants are a potential health hazard because, if present, they may be recognized as antigens by the patient receiving the recombinant protein product.