Transition from 96-well plates to capsules
Figure 6
|
There are three layers of STIC membrane in 96-well format compared with 15 layers in the Nano capsule, which has the same
number as the large-scale STIC devices. An equivalent fold of impurity removal was achieved using Nano capsules compared to
96-well plates with the same process load under identical operating conditions as shown in Figure 6. This might be due to
the fact that interactions between antibodies and STIC ligands vary with solution conditions, not with the number of layers
of membrane or membrane volume. Parameters collected on the 96-well plate can thus be applied to Nano capsule. However, as
reported previously, the antibody loading capacity (or process capacity) is dependent on both solution conditions and membrane
volume or number of layers (24). The DLC should be determined from a small STIC membrane capsule such as Nano or Pico, which
can be directly applied to larger membrane adsorbers.
Removal of HMW species using STIC membrane adsorber
All four model mAbs were derived from stable CHO cell lines and partially purified using MabSelect SuRe resin under current
platform operating conditions. It was reasonable to believe that the host cell protein profiles in these protein A-purified
materials were similar. The highest HCP load was, 578 ppm, or 0.578 mg in 1 g of antibody (in the case of Mab-T). In addition,
residual DNA and leached protein A only accounted for a small portion of STIC binding capacity (data not shown). The STIC
binding sites could not be saturated by residual impurities at a process load of 1 g antibody/mL-STIC. The mechanism behind
the lower HCP clearance during Mab-T STIC purification was therefore investigated.
Since HMW species may, through multiple-site attachment, have greater avidity to the AEX resin or membrane adsorber than the
monomers, AEX in a flowthrough mode was used for HMW removal as previously reported (12, 17). Practically, the removal efficiency
through AEX in a flowthrough mode varies with different antibodies. For some antibodies, HMW can be reduced to a very low
level while in other cases HMW removal is not efficient, and in some extreme cases, HMW reduction is not observed at all.
Thus HMW removal is challenging and should be evaluated for each case. Additionally, HMW removal with AEX resins may be limited
by steric hindrance (25), indicating a potential issue with respect to loading capacity. In the case of membrane chromatography,
the mechanism of mass transfer is convective flow. Therefore, the HMW binding capacity on STIC is expected to be much higher.
Figure 7
|
Figure 7 shows HMW removal from partially purified Mab-T with 1.49% HMW in the load, assessed by SE–HPLC. The HMW in flowthrough
was 0.99% with a load of 2.0 g Mab-T/mL-STIC. The amount of HMW bound to STIC in this experiment was 10 mg. However, as expected,
HCP removal efficiency decreased slightly as the residual HCP in the flowthrough was 40 ppm. In this case, the HMW species
might have stronger interactions with STIC than HCPs. The saturation of binding sites on the membrane adsorber by the HMW
species prevented further removal of trace impurities. However, for the three other antibodies tested, either the HMW level
in the load was low or only minimal HMW removal was observed, and a much higher process capacity was achieved (based on HCP
breakthrough). Thus, caution should be taken if HMW species at an elevated level (>5%) are applied to the membrane chromatography.
A competitive binding analysis of HMW species and other trace impurities should be performed. If STIC offers the same or higher
clearance of HMW compared to other impurities, process capacity might be compromised. Depending on the scale of purification
production, different strategies can be used to mitigate the issue. Membrane chromatography in flowthrough mode with different
mechanisms such as hydrophobic interaction can be incorporated. In addition, multiple cycles of STIC operation can be used
to provide enough manufacturing capacity.
Prediction on large-scale purification production
Figure 8
|
Figure 8 presents a mAb purification production scenario using STIC as an alternative polishing step at large scale. In this
theoretical case, the starting materials are proteins partially purified using a protein A column from 11,000-L HCCFs at a
titer of 5 g/L. The antibody load for STIC is 50 kg. Four cycles of 5-L STIC membrane adsorber operation can provide enough
production capacity for Mab-D, Mab-K and Mab-S. Unfortunately, application of STIC to Mab-T in large scale is predicted to
be challenging due to its lower process capacity or higher residual HCP level. Incorporation of a HMW mitigation step in the
process would be required before being applied to STIC.
Table III
|
The major characteristics of STIC membrane adsorbers are further compared with traditional AEX columns in Table III. A smaller
membrane adsorber device can provide required production capacity, and reduce the plant footprint. As a single-use system,
the STIC membrane adsorber avoids issues experienced in the packing, unpacking, cleaning, and storage of traditional chromatographic
columns. Significant amounts of consumables (e.g., water for injection, buffers, cleaning solutions) are saved and, more importantly,
less related labor is required when membrane chromatography is used. In contrast to the major development effort that AEX
chromatography requires, process development for STIC membrane adsorber is simple and efficient as demonstrated in the previous
sections. Furthermore, the integrity of the membrane adsorber can be assessed using a pre- and post-use filter integrity test
protocol, which is straightforward compared to the HETP test used in traditional chromatographic columns. Lastly, because
of its unique hydrodynamic characteristics, membrane adsorbers can operate at a much shorter residence time or higher operating
flow rate than columns, thus reducing overall processing time and costs. Therefore economic benefits can be achieved using
membrane adsorbers for manufacturing of antibodies as described previously (22).
In summary, STIC provides an alternative to the current AEX polishing step. The two-column production platform can be shortened
by removing the pre-AEX TFF or dilution step. It is extremely valuable for antibodies which have solubility issues at low
ionic strength conditions. In addition, fast screening and optimization followed by process capacity determination in this
article suggests an extremely short development timeline. More importantly STIC can be incorporated into our current platform
in a "plug and play" development approach.
|
