November 1, 2007 (Vol. 27, No. 19)
Sue Pearson Ph.D. Freelance Writer GEN
As Technology Improves, Cost of Goods Goes Down and Titers Go Up
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.
Coming Up On the Outside
The production of some plasmid-based DNA vaccines could also, in the future, increase the demand on the use of microbial-based systems for manufacturing biologics. Simon Thompson, project manager at MMI Group (www.mmigroup.co.uk), discussed promising results with some of the company’s Genvax therapeutic vaccines. The DNA vaccine is based on a plasmid that contains a fusion gene consisting of a targeted antigen and an immunostimulant such as a plant viral gene or a fragment of tetanus toxin.
“Our technology is being evaluated in four Phase I/II trials with DNA vaccines to treat a range of cancers including lymphoma, prostate, colorectal, and lung. In the lymphoma trial we saw that 38 percent of patients that have been analyzed produced an immune response against their own cancer cells,” Thompson stated.
“There are three veterinary DNA vaccines on the market,” Thompson added, “and human DNA vaccines are thought to be not more than five years from coming to market. DNA vaccines can be rapidly manufactured in E. coli, and so when we can produce plasmids at scale, this will be a cost-effective method of producing vaccines that can be rationally designed.”
Insurmountable Challenges?
Speakers at the conference agreed that for microbial cell culture to take off as the dominant manufacturing method for biologicals, there are a few technical problems to overcome. One major challenge for many bacterial systems is the fact that bacteria capable of effective glycosylation are pathogenic, making them a poor choice of vehicle for manufacturing therapeutics.
“Currently,” Dr. Dale stated, “if you want glycosylated proteins using microbial expression systems you can add glycoforms postpurification, but this is so expensive it makes the COGs prohibitively high, so it is not the way to go. Alternatively, engineered yeasts such as Pichia pastoris can make human-like glycoforms, thus probably offering the best route today. There is, however, some excellent research emerging to develop bacterial hybrids where genes from pathogenic bacteria such as Campylobacter jejuni are being introduced into E. coli. If we can get these hybrids working well, then they will offer a truly low-cost alternative for making a much wider range of therapeutic proteins.”
Another issue is one of capacity for plasmid-based DNA therapeutics. “Currently there are only a handful of CMOs worldwide that can manufacture more than 30 grams of plasmid, and few can make kilogram-scale amounts per batch,” noted Hans Huber, Ph.D., technology agent at Boehringer Ingelheim Austria (www.boehringer-ingelheim.com). “Since more biotechs are moving further into the clinic with their DNA vaccines, we will have to have more manufacturing capacity and also improved processes for the large-scale made available.”
According to Dr. Huber, culturing E. coli containing plasmids is not a bottleneck any more as advances in plasmid/host technology and media development mean that plasmid titers of 2 g/L are the benchmark. The problem does, however, lie in getting good quality DNA out of the cells. “The alkaline lysis step designed to release the plasmid DNA from the cell biomass is achieved at the lab scale by mixing, centrifugation, and pipetting, but this is impracticable at large scale as it will cause too much DNA shearing and will increase host cell impurities,” Dr. Huber explained.
“To get around this problem,” he added, “we have designed our own automated alkaline-lysis system containing glass beads and a clarification unit, and using this method we can produce up to 50 kilgrams of plasmid per year, which is sufficient to provide enough DNA vaccine for market supply.”
Delegates at the “European Biopharm Scale-up Congress” agreed that biopharmaceutical production is split between mammalian cell culture and microbial cells, which currently have a 30–40% market share of drug production. The choice of production cell type is driven by drug class. As the industry matures and the protein product arena diversifies, however, it is likely that these lines will blur.
“By 2017,” Dr. Dale concluded, “we’ll routinely see E. coli manufacturing achieving greater than 5 g/L, driven by the need to reduce COGs of biological drugs. As a result of technology advances in antibody design particularly, we will begin to see E. coli, which through 30 years of continuous improvement has always been a good workhorse for manufacturing small proteins, take over from mammalian cells to become the dominant manufacturing vehicle for many protein- and DNA-based drugs.”
