To meet the challenges of supplying a global market with protein biotherapeutics and growing the world market for some therapy classes, the cost of goods (COGs) benchmark has to be less than $100 per gram for manufacturing biologicals, stated Chris Dale, Ph.D., head of microbial technology at Lonza (www.lonza.com), at the recent “European Biopharm Scale-up Congress” in Geneva.
Dr. Dale echoed the sentiments of many speakers at the conference who discussed a range of ways in which manufacturing costs could be significantly reduced by taking advantage of using microbial fermentation as the starting point.
As interest in substituting antibody fragments (Fabs) and single-domain antibodies (nanobodies) for full mAbs increases, microbial expression systems are becoming a popular choice of scale-up vehicle. “In many therapeutic instances you don’t need the Fc region to make a mAb therapy work effectively,” noted Leigh Bowering, Ph.D., senior group leader of microbial fermentation research at UCB Celltech (www.ucb-group.com). “In fact since our Fabs are designed to have an unpaired cysteine, you can attach a PEG molecule to increase the serum half-life, making it similar to a full mAb.”
Dr. Bowering also discussed how UCB Celltech is manufacturing Fab-based therapies in E. coli using a suite of vectors where parameters such as balance of light- and heavy-chain expression and induction can be altered to optimize Fab expression and maximize yield. The process, according to Dr. Bowering, has been scaled up to produce Fab fragments at 1 g/L in a 10,000 liter-scale fermentation with a process that involves a 60°C extraction step, followed by microfiltration or expanded-bed absorption and ion-exchange chromatography.
“Using E. coli to manufacture Fabs, we can get to our first clinical batch rapidly as cell-line and process-development times are only a few months, and we don’t have to use expensive affinity chromatography steps either, so production costs are minimized,” Dr. Bowering concluded.
Laurens Sierkstra, Ph.D., CEO of Netherlands-based BAC (www.bac.nl), agreed with Dr. Bowering that portions of antibodies can be produced much more cost-effectively than mAbs using microbial systems. Dr. Sierkstra presented a case study to show how his company is manufacturing single-domain antibodies in Saccharomyces cerevisiae.
“We tried E. coli, Saccharomyces, Pichia pastoris, and fungi for the expression of our single-domain antibodies,” Dr. Sierkstra explained. “E. coli did not produce a good yield, while Pichia and Saccharomyces did because they secrete the protein. Due to the licensing costs associated with using Pichia, we finally chose to use a Saccharomyces strain.”
According to Dr. Sierkstra, the company has now developed a standard fermentation process using an ethanol feed, which increases yield between 2- and 10-fold, depending on the single-domain antibody, and in 15,000 L can produce yields of 500 mg to 5 g/L. “We can produce single-domain antibodies in just under six months from strain development, so this is a cost-effective and rapid manufacturing process,” noted Dr. Sierkstra.
As well as manufacturing nanobodies and Fab fragments, microbial expression systems can also be used to manufacture peptides. “The market for therapeutic peptides grew from $6.1 billion in 2001 to $14 billion in 2006, and there are more than 400 peptide drug candidates in development, so there is a healthy pipeline,” said Friedrich Nachtmann, Ph.D, head of biotech cooperations at Sandoz (www.sandoz.com).
“Additionally with new administrative routes such as oral and nasal formulations coming online where bioavailability is low and large product quantities are required, cost-effective manufacturing is a must, and this is where using E. coli could be a good way to go,” added Dr. Nachtmann, who presented an autoprotease expression technology called Npro, which works well in E. coli and was jointly developed at Sandoz and the Austrian Centre for Biopharmaceutical Technology.
Npro, an N-terminal autoprotease, is a mutation of a swine fever virus and serves as an expression tag. Npro fusion proteins are accumulated in inclusion bodies, and Npro is able to cleave itself off the target protein exactly behind C168, generating a homogenous N-terminus, reported Dr. Nachtmann. This means that cleavage enzymes do not need to be added, thus saving a process step.
Dr. Nachtmann showed an example where, using the Npro approach, yield of a toxic protein was increased by 40-fold from 0.005 to 0.2 g/L and of a peptide where reduction in COGs was 20–30% at the 3000 L scale and 100% at the 13,000 L scale, when compared to standard fusion protein technology.
“Npro is an excellent method for improving yield of difficult-to-express toxic proteins or peptides in general and offers a good cost-effective alternative to the current fusion protein expression or cytoplasmic expression methods,” Dr. Nachtmann stated.