Combinations/ Antibody-Drug Conjugates

Antibody-Drug Conjugate: Identity

Antibody-Drug Conjugate: Purity

Antibody-Drug Conjugate: Potency

Clinical ADC manufacturing must be executed in an aseptic biological manufacturing environment which operates under cGMP since the antibody modification and cytotoxin conjugation reaction are usually executed under process conditions which will support growth of environmental microbial contaminates. Batch failures resulting from human error or equipment failure are typically low for biological manufacturing.

In the antibody modification suite the naked antibody raw material which is typically frozen at -20°C is thawed and the storage buffer is exchanged via tangential flow filtration. Buffer exchange is often required to alter the pH, salt concentration and remove excipients used to improve storage stability in the bulk antibody solution which may hinder or alter the performance of the conjugation reaction. These steps include the isolator used for weighing and preparation of the solution containing the cytotoxin (Weighing and Dispensing Containment Isolator, Containment Barrier Isolator, and Aseptic Containment Isolator) and  the conjugation suite, in which the cytotoxin is combined stoichiometrically with the antibody linker complex. Bulk ultrafiltration/difiltration (UF/DF) or chromatography is then employed to remove process contaminants such as free cytotoxin and organic solvent.

In the isolator suite cytotoxins can be weighed and solubilized in organic solvent for introduction into the antibody conjugation suite via a hard piped transfer line into the conjugation vessel. The isolator permits safe handling of the cytotoxins in a negative pressure environment and a means of addressing the electrostatic properties of cytotoxins through humidity control. Since the interior of the hood comes in direct contact with potent cytotoxins the hood is designed with a clean in place (CIP) system that permits inactivation of the cytotoxin and/ or multiple detergent and rinse cycles to remove cytotoxin contamination.

In the conjugation suite the coupling between antibody and the cytotoxin is performed in a sealed temperature and pH controlled stainless steel tank with nitrogen over-lay. Special equipment modification in this area such as double mechanical seals, overflow trays and positive displacement pumps with both primary mechanical seals and secondary water seals strengthen the primary containment envelope to protect employees working in the suite. After removal of the process contaminants the buffer containing the ADC is transferred to the formulation suite for transition into the BDS formulation buffer.

All liquid and solid waste from the process are then inactivated and incinerated on site depending on process requirements. Following API concentration adjustment and excipient addition in the formulation suite the BDS is then stored frozen at -20 to -60?C using a controlled rate shell freezer or aseptically dispensed into multiple PETG bottles for bulk storage.

Analytical Support for ADC manufacturing

Analytics are especially challenging for ADCs because they require expertise in both small and large molecule analytical techniques to support in-process, bulk drug substance release, reference standard preparation and drug product stability testing. Reference standard will need to be created, and these methods will include carbohydrate analysis and various methods to assess the level of deamination, oxidation and adducts which may have formed during the production process

Analytical methods selected for testing BDS are designed to assess the targeting ability of the antibody but must also demonstrate the potency of the cytotoxin. Techniques that determine the amount of process and product related contaminates are critical methods for establishing a predicable manufacturing process. Recent audits of project hours have revealed approximately 58% of the total project hours for Technology Transfer are related to developing analytical methods, validation and supporting R&D activities. The same project hour distribution is found with GMP manufacturing in which 55 percent of the total hour are related to QC activities associated with in-process testing and release testing of BDS. Important for multi-product facilities are development of analytical methods which permit detection of cytotoxin in the 10-9 to 10-12 gram range for product change-over. Additional complications such as quality control staff testing raw materials and process samples with free cytotoxin require a lab environment designed to prevent potential exposure to highly potent cytotoxins. Laboratories used for testing will require isolators (Weighing and Dispensing Containment Isolator, Containment Barrier Isolator, and Aseptic Containment Isolator) for sample manipulation and analytical equipment with enclosed auto injectors to prevent exposure to aerosols containing free cytotoxin.

Weighing and Dispensing Containment Isolator (WDCI)

 

Containment Barrier Isolator (CBI) 

 

Aseptic Containment Isolator (ACTI)

Raw Material Supply Chain

ADC(s) have the most extensive supply chain of any biological requiring sourcing of an antibody, linker and potent small molecule cytotoxin. Most biopharmaceutical companied currently developing ADC(s) prefer to use an intact monocolonal antibody targeting agent. The cell bank used to produce the antibody will require extensive virus testing to prove the cell bank is free from adventitious agents. Also the associated downstream purification process for anybody will require evidence that the manufacturing process can inactivate, clear and remove virus if contamination is present. The potent cytotoxins currently used in ADC manufacturing could be semi-synthetic (fermentation derived intermediate) or fully synthetic (chemical synthesis).

Culture Production Factors

A. Cell lines

The great majority of commercial mAbs are produced in Chinese hamster ovary (CHO) and NS0 cells, originating from plasmacytoma cells that were modified until IgG generation in non-secreting B cells.

Microorganisms modified by genetic engineering techniques have attracted much focus since these cells are simpler to handle and easier to modify compared to animal cells. Other advantages of production methods using genetically modified organisms are that these cell have well-defined expression systems, and the production methodology is reproducible and easy to validate. Modified yeast cells, such as Pichia pastoris have a great potential for usage since these cells are known to achieve high secretion levels of heterologous proteins.

B. Culture Medium

Cultivation media for mammalian cells must have a complex content of ingredients ranging from amino acids to trace elements. To supply the cellular demand of these nutrients, the culture medium uses serum.

Super Plus™ is a cell culture supplement designed to reduce or eliminate the requirement for serum in the basal medium of cell culture. Super Plus™ also minimizes culture variance due to inconsistent quality between serum batches. Super Plus™-added medium supports growth of many cell lines, including MDCK, VERO, BHK, ST, PK-15, HEK-293, and CHO.

Plus™ VERO is an animal component-free culture medium developed specifically for VERO cell culture.

Plus™ MDCK is an animal component-free culture medium developed specifically for MDCK cell culture.

C. Culture Conditions

Growing conditions can directly influence the cell growth and production levels of molecules of interest. Usually, mammalian cell culture conditions for mAb production are very well defined: 37° C, pH 7.15, and dissolved O2 (OD) levels at 30-60%.

D. Production Platforms

      I. Batch Production

The simplest of all the production platform is the batch production, which consists of a closed system were a bioreactor is sterilized and prepared with a medium containing all the nutrients needed for cellular growth and product manufacturing.

      II. Continuous Fermentation

There are two types of continuous production: chemostat cultures and perfusion cultures. For chemostat cultures, fresh medium is added to the bioreactor and fermented medium is removed along with cells at a constant flow rate so that the culture volume remains unchanged. The flow rate controls cellular growth and when these two variables are equal, the bioreactor reaches equilibrium-cell concentration, nutrient concentration, and product concentration are held constant. To avoid viable cell loss along with constant outflow of the by-products of cell metabolism, many manufacturing plants have developed a cell-recycling system and thus, the perfusion method was developed where cells are kept inside the bioreactor.

      III. Fed-batch processes.

This is by far the most utilized at industrial scale. In this process, the cell density reaches 8-12 x 10 6 cells/mL, and cell viability in the bioreactor is enhanced by controlled nutrient addition at specified intervals.

E. Production Systems

  1. Production Systems for cells in suspension cultures

Stainless steel stirred tank bioreactors are the most consolidated type of bioreactor used for industrial mAb production and consist of baffle-stirred tanks linked to rotor systems. It is a consolidated system, and there is a lot of knowledge and experience surrounding this technology, acquired by its vast industrial use beyond production using mammalian cells.

StirCradle is a desk-type bioreactor/fermenter for microorganism culture. By combining the vessel, workbench, controller and peristaltic pumps into one integrated system, this patented system effectively provides the most capacity for a small footprint. The StirCradle is ideal for research and development involving microorganism or animal and plant cell cultures.

The StirCradle-Pro is a fully stainless steel fermenter/bioreactor system designed with an automated 5-step cycle SIP system for both culture medium and reactor vessel. VacciXcell offers a wide range of standard reactor capacities from 20L up to 1000L; however, customized reactor sizes are available should the users require.

Air lift reactors are also broadly used for the industrial production of mAbs. The reactor consists of tanks with a bubble column inside, and air is injected into the column base. The air flows through the column’s length to the top of the bioreactor as degassed culture medium flows in the opposite direction to the reactor bottom. This creates a constant gentle mixing of the medium as well as proper culture aeration, annulling part of the shear stress caused by other stirring systems.

Disposable bioreactors bought many changes for mAb manufacturing. At the end of the process, the bioreactor is discarded and replaced by a new clean and sterile one. This eradicated cross contamination between batches and decreases the time consumed with the equipment preparation between batches.

CelCradle™ is a disposable bioreactor capable of high-density cell culture for protein expression, virus, and monoclonal antibody production. It is designed based on the concept of bellow-induced intermittent flow of media and air through porous matrices, where cells reside. This provides a low shear, high aeration, and foam-free culture environment.