Concentration and Buffer Exchange
Because of the relatively large size of the VLP molecules, a high molecular weight cut off membrane such as 500 or 1,000 kDa
may be used for this application. The large molecular weight cut off has the advantage of reducing the host cell protein (HCP)
content while recovering the VLPs in the retentate fraction.
Anion Exchange Chromatography
An effective approach for the reduction of DNA and endotoxin levels is the use of AEX. Owing to the shear size of the particles,
there are inherent mass transfer limitations that play a significant role during any chromatographic step. This reduces mass
diffusion during column loading, reducing the effectiveness of the step and limiting of the column's overall dynamic binding
capacity. Scale may then become an issue. There are several technologies available to negate this problem such as membrane
adsorbtion technology, available from a number of suppliers such as Millipore, Pall, and Natrix Separations. Eden Biodesign
has found the Poros HQ diffusion chromatography media (Applied Biosystems) to be effective. A typical chromatogram obtained
during an anion exchange purification of a VLP is shown in Figure 4.
Figure 4. Anion exchange chromatogram for the purification of virus like particles (VLPs)
Size Exclusion Chromatography
Taking the platform approach, the size of the particles should be in the range of 30–50 nm. This is above the molecular exclusion
limit for a number of size exclusion resins, and therefore, they may be used in a group separations mode whereby the large
VLPs are above the upper exclusion limit for the resin to pass through in the void volume. The smaller HCPs and misformed
VLPs pass through into the pores of the resin and are retained on the column for longer, eluting with a greater residence
A typical group separations chromatogram is shown in Figure 5, peak one in the void volume being the VLPs and peak two being
gross impurities such as residual HCPs and misformed particles.
Figure 5. Size exclusion chromatogram for the purification of virus-like particles (VLPs)
There are a number of problems associated with the production of therapeutic products and vaccines from microbial systems,
specifically from E. coli. These are centred mainly on the primary separations and reduction of lipopolysaccharides (LPS) from the process stream.
A major challenge in the production of any therapeutic products from E. coli is the requirement for the removal of LPS from the process stream, a specific processing step for this application may be
required. LPS or endotoxins are a major component of gram negative bacteria such as E. coli. Endotoxins, even at relatively low doses can initiate an innate immune response resulting in a fever and in severe cases,
septic shock and even death. Regulatory specifications exist for many parenteral products and are typically 2 IU per dose
or less. There are many technologies specifically for this application such as LPS affinity chromatography and membrane adsorbtion
technology. Membranes are a more attractive proposition because they are cheap, simple to operate, and require little or no
cleaning validation. There are several options available for this application such as Pall's Mustang E and Sartorius's Q membrane.
Membranes such as the Sartobind Q have a slightly different chemistry to the Mustang E but work on a similar charge effect,
with the added advantage of a 3 μm pore size, so that few or no losses are seen because of the size exclusion effect that
may be observed when processing such large molecules as VLPs.
However, this is not the complete picture; mechanically removing free endotoxins from the process stream is only half the
challenge. Given the typically hydrophobic nature of VLPs, there is the additional problem of endotoxins adhering to the particles
themselves, offering additional and significant further challenges to reduction. Bound endotoxins may be released by treatment
with solvents, detergents, or a combination of both, further complicating the manufacturing process and adding expense. A
combination of endotoxin reduction techniques is the more favourable approach. A production strategy in yeast such as Pichia pastoris may also be the favoured approach to avoid this problem.