Fermentation technology: cooking up new therapeutics

With a new state-of-the-art, cGMP fermentation facility in Jerusalem, SAFC is looking at new applications for what is viewed by many as a traditional technology. These applications include the synthesis of High Potency Active Pharmaceutical Ingredients (HPAPIs) and therapeutic drugs.

Therapeutic drugs are substances used for health restoration in the treatment of disease, drawing on the utilization of microbial bio-factories. These bio-factories offer the pharmaceutical industry a reliable, powerful and economic source for therapeutics. In order to demonstrate the diversity of applications for bio-factories, Silvian Shama, business development manager at the Jerusalem plant, looks at examples of therapeutic drugs that are primarily isolated through microbial fermentation.

chemical therapeutics

The term chemical therapeutics refers to toxic small organic molecules with a molecular weight of 1000-2000 which are produced naturally by micro-organisms in response to stress and are called secondary metabolites. These molecules may be complex in nature, such as heterocyclics, with several chiral centres, large stereospecific rings or a unique conjugated double bond system. Small, cytotoxic molecules are employed in anti-cancer therapy (chemotherapy) and also in the treatment of infectious diseases (antibiotics).

Cancer therapy - The anti-cancer therapeutic activity of small molecule drugs in clinical use is limited by their general toxicity to proliferating cells, including some normal cells. In the lack of specificity and effective targeting, chemotherapy using High Potency Active Pharmaceutical Ingredients (HPAPIs) involves high doses and often severe side effects.

Over the last few years the pharmaceutical industry has taken a new approach to targeted drug delivery by conjugating HPAPIs to monoclonal antibodies creating highly specific ‘smart bombs’ that can be directed to cancer cells through highly expressed antigens. This means that not only are smaller quantities of a cytotoxic drug required, but non-target cells are spared many of the cytotoxic effects. Various HPAPI Antibody Drug Conjugates (ADCs) are currently in clinical trials with major pharmaceutical companies and some are gaining FDA approval for difficult-to-treat indications, mainly in the treatment of cancer.

Example 1

Wyeth’s FDA-approved Mylotarg ADC consists of a recombinant humanised IgG4 kappa antibody, conjugated with a cytotoxic anti-tumor antibiotic, calicheamicin, which is isolated through the fermentation of a bacterium, Micromonospora echinospora subspecies calichensis. The antibody portion of Mylotarg binds specifically to the CD33 antigen, a sialic acid-dependent adhesion protein found on the surface of leukemic blasts and immature normal cells of myelomonocytic lineage, but not on normal hematopoietic stem cells.

Example 2

Genentech’s trastuzumab-DM1 is an ADC consisting of ImmunoGen’s DM1 agent linked to Genentech’s humanised anti-HER2 antibody, trastuzumab (Herceptin, approved to treat breast cancer). DM1 is a proprietary derivative of a naturally occurring substance belonging to the Ansamycin family of secondary metabolites and is isolated through fermentation of an actinomyces species - Actinosynnema pretiosum.

Treatment of infectious diseases - Antibiotics are substances or compounds that kill or inhibit the growth of micro-organisms, mainly bacteria (bactericidal or bacteriostatic agents, respectively).

Many of the antibiotics are sourced from living micro-organisms, used either in their natural state or in the case of semi-synthesis, chemically modified. Semi–synthesis is usually used when the precursor molecule is too structurally complex, too costly or too inefficient to be produced by total synthesis. It is also possible that the semi synthetic derivative exhibits better performance than the original biomolecule itself in terms of potency, stability or safety.

Example 1 – Aminoglycosides

Aminoglycosides are produced and isolated from bacteria of the Streptomyces genus, used in their existing form to provide drugs such as Streptomycin, a bactericidal agent and Hygromycin B, a killer of bacteria and fungi.

Example 2 – Doxycycline

Doxycycline is a semi-synthetic tetracycline. Tetracyclines were isolated from Streptomyces subspecies such as aureofaciens and rimosus. Doxycyclin was developed in the ‘60s by Pfizer and marketed under the brand name Vibramycin.

Biological therapeutics

The term biological therapeutics refers to large organic molecules, mainly proteins. Proteins are polymers of amino acids arranged in a linear chain and folded into a globular form. The sequence of an amino acids protein is defined by the sequence of a gene, which is encoded in the genetic code. Biological therapeutics are isolated from the native producing micro-organisms or, more often, expressed in a heterologous microorganism using recombinant DNA technology (recombinant proteins). Fermentation derived biological therapeutics have been employed in a wide range of medical indications.

Native protein example – Collagenases

Collagenases are a group of enzymes produced by the bacterial pathogen Clostridium genus. Upon infection, Collagenases break the collagen peptide bond, a key component of the animal extracellular matrix. Destruction of the extracellular structures causes severe tissue damage and can facilitate the spread of serious medical conditions such as gaseous gangrene.

Essentially turning an enemy into a friend, native isolated collagenases are employed in a variety of medical indications involving collagen disorders.

Collagenases approved for medical uses are: Santyl ointment and Iruxol, for the removal of dead skin from wounds and burned areas in poorly healing wounds and necroses, and Xiafles Dupuytren’s contracture, a condition resulting in the contracture of the fingers into the palm. Other collagenases are in currently in clinical trials for indications such as:

• Frozen shoulder - an inflammation and thickening of the shoulder capsule;
• Cellulite - a condition characterised by dimpling of the skin typically affecting the thighs and buttocks; and
• Lipomas - benign fatty tumors that occur as bulges under the skin.

Recombinant proteins - Are manufactured in various heterologous microbial hosts, mainly bacteria, including E.coli, Pseudomonas fluorescens and Yeast (Pichia pestoris). Recombinant proteins have various advantages over native proteins such as high yields, defined reproducible product, expression of humanised proteins (which avoid the immunogenic complications associated with non-human proteins) and the possibility to improve activity by gaining mutations.

Example 1 – Lysostaphin

Lysostaphin is an endopeptidase produced naturally by Staphylococcus simulans. Specifically, Lysostaphin degrades the cell wall peptidoglycan of Staphylococcus aureus.

With the isolation of clinical strains of Staphylococcus aureus carrying the gene that confers antibiotic resistance, the need for novel antistaphylococcals has become increasingly urgent.

Recombinant Lysostaphin has been investigated in various models for infections caused by an S. aureus, either systemic, or specific, such as endocarditis (inflammation of the inner layer of the heart).

Example 2 – Fibroblast Growth Factor

Growth factors are signaling agents that induce cellular growth by promoting differentiation or proliferation. Basic Fibroblast Growth Factor (bFGF) possesses broad mitogenic and cell survival activities.

Recombinant FGF has been implemented in various disorders including angiogenesis in limb ischemia, tissue regeneration, cartilage injuries, wound healing and remodeling of bone and cartilage.

Example 3 – Antibody fragments

In recent years, it has been recognised that the use of full-length antibodies may not always be necessary or even desirable. Antibody fragments are small portions of the full length antibody, sufficient to confer therapeutic activity, for example FAB (fragment antigen binding) fragments, containing variable heavy and light chains whose small size allows enhanced issue penetration, particularly in solid tumors. Fragment production with microbial systems has been demonstrated with E. coli, yeasts, and fungi.

Various companies have antibody fragments under development including Ablynx single domain nanobodies which are being tested for cardiovascular, Inflammatory, pulmonary and neurology diseases, and in oncology. Some approved products are already on the market such as Genentech’s Lucentis (ranibizumab injection), which is a recombinant humanised IgG1 kappa isotype monoclonal antibody fragment designed for intraocular use.

Applications of microbial fermentation technology in new therapeutics are expanding and SAFC offers unique cGMP fermentation-based manufacturing capabilities through its Jerusalem facility, including high potent APIs and biologics. SAFC scientific teams provide a complete range of services, including process evaluation and development, optimisation and scale up, technology transfer, manufacturing, analytical testing and regulatory filing to help customers bring new drugs to market faster.