We produce peptides
efficiently and predictably
in E. coli

NUMAFERM’s platform technology

Available biotechnologies can be applied for the production of many enzymes (and other proteins). Batch sizes in the tons scale are common and the production costs can be as low as 10-20 €/kg (van Beilen et al., Current opinion of biotechnology, 2002). Available biotechnologies are, however, limited towards the recombinant production of peptides. This is due to three major bottlenecks: 1. omnipresence of proteases and the resulting degradation of peptides (figure 1), 2. aggregation of peptides by mainly hydrophobic interactions complicating downstreaming processes (DSP) and 3. toxic effects of peptides, especially antimicrobial peptides, towards the production host. NUMAFERM’s technology copes with these challenges and serves as a reliable and cost-efficient recombinant production platform. How have we achieved this success? We have developed bio-based process innovations to produce peptides predictably, effectively and cost-efficiently.

Secretion Approach

We have commercialized the first secretion technology for Gram-negative bacteria; our working horses are common E. coli strains. In addition, we have developed a trifunctional fusion partner with fascinating characteristics enabling recombinant peptide production – the NUMAtag. How does it work? In a first step, requested peptides are fused with the NUMAtag. The NUMAtag is recognized by a so-called Type 1 secretion system (T1SS) that catalyzes the one-step transport of the fusion peptide across the Gram-negative cell wall into the protease-free supernanant of E. coli cells (tag functionality 1: TRANSPORT).

This transport across two phospholipid membranes is called secretion (figure 2). This protects the peptides from being destroyed by proteases.  Type 1 secretion systems (T1SS) consist of three different membrane proteins that form an ATP-driven transport nanomachinery.

After transport, the NUMAtag remains attached to the peptide and increases the solubility of the peptide (tag functionality 2: SOLUBILITY).
Moreover, the NUMAtag sterically prevents the interaction of peptides with the production host and therefore decreases cytotoxic effects. This is especially important for the production of antimicrobial peptides (functionality 3: PROTECTION). Taken together, these functionalities enable high production titers (decreased toxicity) of intact peptides (no proteases present) and facilitates the downstreaming processing (increased solubility).
As side effect, the initial purity of fusion peptides is very high (see lane a in figure 2), which enables cost-efficient downstreaming processing (DSP). In many applications the NUMAtag remains attached to peptides as it does not influence requested functionality negatively. If requested, the NUMAtag can be efficiently removed and 100% sequence-identical peptides can be produced. This is particularly important for pharmaceutical applications.

By those means, NUMAFERM’s biotechnological production platform solves limitations of available technologies and enables the cost-efficient recombinant manufacturing of peptides.

In addition to that we established another smart solution to produce peptides. We call this approach the NUMAswitch. It enables the production of “Soluble Inclusion Bodies” and handles the major limitation of classical inclusion body-tags.

Molecular Switch 

In a first step, requested peptides are fused with the NUMAswitch. This bifunctional switch transfers two functionalities toward the peptide.
First, peptides are forced to form insoluble aggregates insides the cell, called inclusion bodies (functionality 1: INCLUSIONBODY, IBtag; OFF-state of the switch). These aggregates are resistant towards proteases and fusion peptides are not degraded. Expression levels of peptides fused to the NUMAswitch are very high and standardized high-cell-density fermentation protocols to increase product titers are well-established at NUMAFERM. Normally, there is one major limitation with conventional IBtags: the conversion of the aggregates to soluble fusion peptides (called renaturation). And this makes sense, since the purpose of IBtags is to form aggregates rather than soluble fusion peptides. However, soluble fusion peptides are in many cases necessary.

Because that state will allow obtaining a functional fusion construct or to cleave of the NUMAswitch with a site specific protease. Currently, this is a serious hurdle since either renaturation does not work at all or it is very inefficient (≤ 25% yield). The NUMAswitch offers the solution to that issue and fusion peptides can efficiently be renatured (functionality 2: RENATURATION). After the inclusion bodies of requested peptides fused to the NUMAswitch are solubilized (in chaotropic salts, detergents, organic solvents, bases etc.), they are incubated with NUMAFERM’s proprietary renaturation buffer. The construct is then renatured highly efficient (close to 100% yield) with concentrations in the mg/mL range (ON-state of the switch, see Figure 3, step 3). This enables the utilization of functional, soluble fusion peptides or the application of site-specific proteases (which are non-functional in the environment of solubilization reagents described above) to cleave-off the peptide.

Altogether, the NUMAswitch offers the advantages of IBtags (protection of peptides from proteolysis, high-level expression, good initial purity), solves existent limitations (inefficient renaturation) and serves as reliable, cost-efficient, recombinant production platform for pure peptides aswell as functional fusion peptides.

Advantages of our technology

Peptides become much cheaper with our technology. Especially when it comes to larger amounts and longer sequences, our technology holds significant advantages in production costs.

We can significantly reduce the carbon footprint. The production of 1 kilogram may require more than 25 tons of raw materials of which some a very toxic. Our bioprocess only requires a few hundret kilograms on non-toxic feedstock.

We can produce peptides of any lengths within a week. In chemical synthesis, this process may take up to several months, depending on the length of the peptide.

We can easily produce long peptides. The limitation in peptide length is one of the major drawbacks of chemical synthesis, making them overproportionally expensive with every added amino acid.

FAQ

What is the maximum length of a peptide you can produce?

There is literally no limit in peptide length in regards to our production technology. We can produce both, oligopeptides and polypeptides. Peptides are typically defined to consist of 2 to 100 amino acids. Our technology can even go beyond this size and produce small proteins or enzymes.

How do I order peptides at NUMAFERM?

Simply give us a call, send an email to info@numaferm.com or use our contact form. Looking at the desired sequence, we can quickly give you a statement on whether we see our technology capable of producing the peptide.

Which purity levels do you offer?

All our products have a purity of >85% and we can easily move it to >99%. We do not offer anything below as our production technology grants a very high initial purity.

Do you produce under GMP conditions?

We realize GMP production with partners. Our technology is generally suitable for GMP production. GMP-like material for clinical studies and filings can be produced within typically required time frames.

Is it possible to produce c-terminus amidated peptides?

No, this is not possible.

Do your products have glycolizations?

No, our products are completely free of glycolizations.