Polishing Complex Therapeutic Proteins - A new downstream purification platform using a salt-tolerant membrane adsorber. - BioPharm International


Polishing Complex Therapeutic Proteins
A new downstream purification platform using a salt-tolerant membrane adsorber.

BioPharm International
Volume 26, Issue 1, pp. 42-51

Sartobind STIC has greater binding strength than Sartobind Q

Figure 1: Schematic comparison of three generations of purification processes for non-mAb recombinant therapeutic proteins at Bayer. a) Older generation purification process. b) Current platform purification process. c) Future platform purification process (i.e., STIC process). A is affinity, FR is blast freeze, IA is immunoaffinity, IE is ion exchange, MA is membrane adsorber, VI is viral inactivation, VF is viral filtration, UF is ultrafiltration, and DF is diafiltration. (ALL FIGURES ARE COURTESY OF THE AUTHORS)
The first step was evaluating the level of salt tolerance of Sartobind STIC in comparison with Sartobind Q (see Figure 2a). Purified Bay-A001, a Bayer recombinant protein product, was loaded to either Sartobind Q or Sartobind STIC under neutral pH and low salt concentration so that the protein binds to the membranes. A step-wise NaCl gradient wash was conducted to determine the NaCl concentration necessary to elute Bay-A001 from each MA. Some split peaks and conductivity fluctuations were observed, which were probably caused by nonideal flow distribution inside the LP15 membrane holder. Running the same gradient through the bypass line did not produce any conductivity fluctuation (data not shown). This flow distribution issue, however, did not affect the interpretation of the results. The elution of Bay-A001 from Sartobind Q started in the 0.2 M NaCl fraction and continued in the 0.3 and 0.4 M NaCl fractions. This product was inherently heterogeneous and the elution into multiple fractions as shown in the study was consistent with what was seen before. In comparison, no significant elution was observed from Sartobind STIC at NaCl concentrations up to 0.6 M. An elution peak was observed in the last fraction with 1 M NaCl, although the integrated peak area was significantly smaller than the total integrated peak areas from the Sartobind Q chromatogram. Because the same amount of protein was loaded to each MA, the data indicated that not all protein was eluted from Sartobind STIC at 1 M NaCl. Overall, this experiment demonstrated the salt tolerance of Sartobind STIC. It also showed that a high salt concentration was needed to ensure good product recovery for processing proteins, such as Bay-A001, in a flow-through mode using Sartobind STIC.

Sartobind STIC as a polishing step for high-salt immunoaffinity eluate

Figure 2: a) Step-wise salt elution of Bay-A001 from Sartobind Q and Sartobind STIC. Brown line is conductivity trace, red line is A280 trace from Sartobind Q, and blue line is A280 trace from Sartobind STIC. Numbers at the bottom are molar concentrations of NaCl in different fractions. b) Step-wise elution of Bay-A001 from Sartobind STIC using increasing percentage of immunoaffinity elution buffer. Brown line is conductivity trace and blue line is A280 trace. Numbers at bottom are percentage points of immunoaffinity elution buffer in different fractions.
The possible implementation of Sartobind STIC into the platform purification process was evaluated. One option was to use Sartobind STIC to replace the current Q MA polishing step, which served solely as a DNA removal step without the requirement for any feed stream adjustment. The drawback, however, was that a salt addition into the feed stream would be needed to ensure good recovery from Sartobind STIC. This added operation contradicted the goal of process improvement. The only step in the process where the salt concentration was high enough for Sartobind STIC flow-through operation was the immunoaffinity eluate. It was therefore decided to use Sartobind STIC to polish the immunoaffinity eluate in flow-through mode to remove residual DNA and HCP. The salt tolerance feature of Sartobind STIC meant that less dilution of the immunoaffinity eluate was needed. The impurity clearance performance by Sartobind STIC would decide whether any other polishing steps were required.

Figure 3: Dynamic binding capacity of host cell impurities by Sartobind STIC. a) Host cell proteins breakthrough curve with host cell protein (HCP) spike-in. b) DNA breakthrough curve with representative immunoaffinity eluate.
An important parameter for the proposed Sartobind STIC operation was the maximum dilution on the immunoaffinity eluate, which should give a salt condition high enough to give good product recovery, while maximizing impurity removal. To determine the dilution target, purified Bay-A001 was loaded to a Sartobind STIC LP15 device at low salt concentration. The membrane was then washed with a 0–50% step-wise gradient of the immunoaffinity elution buffer (see Figure 2b). The chromatogram showed that with 30% immunoaffinity elution buffer, almost all the proteins were eluted. The conductivity of the 30% immuno-affinity elution buffer solution was measured to be 39 mS/cm at 5 C.

Table I: Process parameters of Sartobind STIC unit operation in a laboratory-scale STIC process for Bay-A001.
The authors then investigated whether Sartobind STIC could clear DNA and HCP under the same buffer conditions. HCP spike-in for HCP clearance evaluation was chosen because the low HCP level in the immunoaffinity eluate and its high salt content prevented the authors from getting reliable assay results. A small aliquot of immunoaffinity load material, which had HCP as the major protein content, was spiked into a diluted immunoaffinity elution buffer at 39 mS/cm at 5 C. The solution was loaded to a Sartobind STIC LP 15 device and flow-through fractions were collected. Interestingly, the HCP assay showed an immediate 5–7% breakthrough (see Figure 3a), which remained steady at HCP load densities up to 800 g/mL membrane. Because HCP consisted of a mix of proteins, it was likely that some of the more basic proteins did not bind to Sartobind STIC under the testing conditions, causing immediate breakthrough. Nevertheless, HCP clearance observed in this experiment was significant and possibly sufficient to reduce HCP to a level within the acceptable range for the Bay-A001 drug substance. For the evaluation of DNA clearance, Bay-A001 immunoaffinity eluate was diluted to 39 mS/cm at 5 C and loaded to a Sartobind STIC LP15 device. Flow-through fractions were collected and the DNA content in load and flow-through fractions was analyzed by quantitative polymerase chain reaction (qPCR). As shown in Figure 3b, no DNA can be detected in the flow-through at DNA load densities up to 45 g/mL membrane volume. The DNA load density was limited by the availability of feed material and the maximum DNA binding capacity was expected to be much higher. A DNA binding capacity of 24 g/L for Sartobind STIC at 16.7 mS/cm was previously reported (17). Overall, it appeared that by diluting Bay-A001 immunoaffinity eluate to 39 mS/cm at 5 C, Sartobind STIC in flow-through mode could be used to remove DNA and HCP with good product recovery. Although the maximum impurity load densities achieved in these studies were not high, they still represented good process throughput because the impurity concentrations in immunoaffinity eluate were very low.

Table II: Critical quality attributes of drug substances (DS) from Bay-A001 laboratory-scale STIC process are comparable to those from current process at manufacturing scale. N.D. = not detected; NA = not available. SEC–HPLC is size-exclusion chromatography–high-performance liquid chromatography, SDS-PAGE is sodium dodecyl sulfate polyacrylamide gel electrophoresis.
With its DNA and HCP clearance capability from immunoaffinity eluate, Sartobind STIC has the potential to replace polishing columns and the Q MA polishing step in the platform process. For the new platform process, which was tentatively called STIC process, there are only three chromatography unit operations: a Q MA capturing step, an immunoaffinity column, and a Sartobind STIC flow-through polishing step (see Figure 1c). To determine whether STIC process could produce Bay-A001 drug substance comparable to the current platform process, three laboratory-scale purification trains using STIC process were performed. Viral filtration was not performed in the laboratory-scale runs. Based on previous experience, viral filtration can reduce HCP and aggregates in Bay-A001 by approximately two-fold. The process parameters for the laboratory-scale Sartobind STIC step are listed in Table I. Table II compares the performance of laboratory-scale STIC process with that of the current platform process at manufacturing scale. Sartobind STIC has an average step yield of 93%. The combined yield of the three polishing steps that Sartobind STIC replaced is 92%. Thus, no difference in overall yield was expected between the two processes.

Figure 4: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of Bay-A001 drug substances generated from laboratory-scale STIC process. a) Commassie blue staining. From left to right: Lane 1: Bay-A001 reference standard, Lane 2: lab scale run 1, Lane 3: lab scale run 2, Lane 4: lab scale run 2, Lane 5: lab scale run 3, Lane 6: lab scale run 3, Lane 7: Bay-A001 reference standard. b) Silver staining, left lane: Bay-A001 reference standard, right lane: laboratory-scale run 1,. c) Western blot, left lane: Bay-A001 reference standard, right lane: laboratory-scale run 1.
Critical quality attributes of the drug substances from the two processes were also compared. HCP levels from STIC process are low and comparable to those from the current process, even without the additional clearance from viral filtration. Two STIC process runs had DS DNA levels below detection, as is the case with the current process. The other STIC process run had DS DNA at 2.8 pg/dose, significantly lower than the specification for this product and regulatory guidelines. Aggregates were about two-fold higher in DS from STIC process compared with the current process. However, aggregate levels were expected to be comparable if viral filtration is included in the STIC process based on our experience that the viral filter provides about two-fold reduction in aggregates. Degradation product levels tested slightly higher in STIC run 2 and 3, but were well within acceptable range and should not be a quality concern. Purities as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Coomassie blue staining were comparable between the two processes. Specific activity was somewhat lower with STIC process, but still within the acceptable range. Data analysis showed that the lower specific activities were correlated to lower step yields at the laboratory scale UF/DF and not directly caused by STIC, which could be an equipment-specific issue. More extensive comparability studies at a larger purification scale following this proof-of-concept will show whether this difference is consistently obtained. The immunoaffinity column does not have significant ligand leaching based on previous experience. Thus, the IgG clearance capability of Sartobind STIC was not characterized. For both processes, the levels of mouse IgG in drug substance were below the assay's limit of detection. Figure 4 shows the SDS-PAGE analysis, including Coomassie blue staining, silver staining, and Western blotting, of STIC process drug substance in comparison with Bay-A001 reference standard. No unknown band or significant change in band pattern was observed in STIC process drug substance. Overall, the STIC process reduced the number of unit operation by two compared with the current platform process, without sacrificing yield or product quality.

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