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Comparison of the integrated assembly purified MGG to the control revealed that the purity if the MGG was very high.
A team assessed the means to improve the production process of a polyclonal antibody from mouse serum at the 200-L production scale. The objectives of the study were to improve the current process by reducing processing time, increasing yield and purity, and decreasing operation costs. An integrated disposable assembly was built, incorporating disposable technologies for:
1. Clarification and bioburden reduction.
2. Affinity purification (using a prepacked column).
3. Concentration and diafiltration.
4. Final filtration (using an in-line filter connected to a bag).
The solution reduced labor costs, accelerated turnaround process time, and ensured high purity. This study was a successful first step; the proposed next step is an assessment of changes to be implemented at process scale, specifically to implement tangential flow filtration (TFF) for concentration, and to replace the final dialysis step with diafiltration.
The increasing demands on productivity in biologics processing have led companies to seek out process efficiency more broadly instead of considering only yield. Manufacturers now seek to improve batch and campaign changeover time to allow more flexible multiproduct operations. This change in views has led to increased demand for disposable technologies, which significantly reduce the changeover time between batches or campaigns. This increased demand for disposable technologies applies to the manufacture of biologicals used in diagnostics and biotherapeutics. In this study, we evaluated disposable technologies throughout a mouse gamma globulin (MGG) process to improve overall productivity in the manufacture of this diagnostic.
The current method used to produce MGG is a batch approach that is described below. For clarification and bioburden reduction, glass fiber–poly-propylene capsules (1–1.2 μm) were used in prefiltration. Intermediate filtration was conducted using polypropylene–cellulose acetate–polyethersulfone capsules (0.65–0.45 μm). Bioburden reduction was performed using cellulose acetate–polyethersulfone capsules (0.2 μm).
Chromatography media (ProSep-vA Ultra, 5 L) at a dynamic binding capacity of 20 mg/mL was used for the chromatography step. Two hundred liters of media were processed in a batch-wise manner with a target step yield of 90 g and 95% purity. The following buffers were used for the bind, wash, and elute steps:
1. Binding buffer: PBS 0.01 M, NaCl 0.15 M, azide 0.1%, pH 7.1
2. Wash buffer: PBS 0.01 M, NaCl 0.15 M, azide 0.1%, pH 7.1
3. Elution buffer: glycine HCL 0.2 M, azide 0.1%, pH 2.8
4. Neutralization buffer: tris 0.5 M, NaCl 0.5 M, azide 0.1%, pH 10.2
5. Cleaning: acetic acid 2 M, ethanol 20%, after 300 L.
For concentration and dialysis, a stirred cell concentrator with a 30 kD molecular weight cut-off (MWCO) PM membrane or a 20 kD MWCO cellulose acetate membrane were used to concentrate purified MGG to at least 35–60 mg/mL. Dialysis was performed using tubing (10–14 kD MWCO) at 2.8 °C with PBS 0.1 M, NaCl 1.5 M, and azide 0.1%, pH 7.1 for at least 24 hours with three changes of dialysis buffer. Final filtration was performed using a double membrane filter (0.8/0.2 μm). Final filtration though a 0.2-μm filter was conducted on the same day as the fill-and-finish operation. The target concentration was 25–50 mg/mL with a minimum purity of 95%.
Described here is an experimental approach to improve the purification of MGG from serum at a 200-L production scale. The objective was to purify mouse MGG with an integrated disposable assembly at a 3% scale that balanced availability of feedstock, and to provide an appropriate scale of operation to accurately assess the scaled-down operation.
The integrated disposable assembly (Figure 1) was built using neoprene tubing (Pharmed, Miami, FL) of suitable lengths and with the appropriate connectors attached. These were placed in autoclave bags and sterilized at 121 °C for 20 minutes. The filters were aseptically connected to tubing assemblies in a laminar flow hood.
Chromatography buffers were sterile filtered in bags using 0.2 μm filters (Opticap XL3) with Millipore Express SHF membrane in the following quantities: equilibration buffer: 50 L; elution buffer: 10 L; and regeneration buffer: 50 L. The bags were aseptically connected to the assembly as shown in Figure 1.
A 32 x 250 mm column (Millipore Vantage, Billerica, MA) was manually packed offline with chromatography media (ProSep-vA Ultra, 200 mL) in 20% benzyl alcohol. Packing linear flow velocities were 1,000 cm/hr. Before applying the feedstock, the column was washed with 10 CV of elution buffer, followed by 10 CV of binding buffer. The column was then aseptically connected to the disposable assembly as shown in Figure 1.
The TFF system was assembled, and the 30 kD membrane (Pellicon 3, 0.11 m2 ) was rinsed with 10 L of sterile water (Milli-Q) to remove any preservatives. The system was sanitized with 5 L of 0.5 M NaOH. After sanitization, the assembly was rinsed with sterile water until a neutral effluent pH was measured from the cross flow and permeate lines. The assembly was then aseptically connected to the eluate bag as shown in Figure 1.
Frozen mouse serum (6 L) was thawed then pumped into the disposable assembly. The flow rate was slowly ramped up to an inlet pressure of 10 psi. All of the serum was allowed to pass first through the clarification filter (Opticap XL5 capsule with Polysep II 1.0/0.2 μm cartridge), followed by the sterile filter (Millipak 200 gamma gold 0.22 μm filter) into a 10-L bag. The clarification filter was changed twice because of by-plugging.
The serum was processed in a continuous operation in the assembly. A volume of 800 mL was pumped onto the column (ProSep) at a flow rate of 60 mL/minute. The void volume for the tubing and column was found to be approximately 100 mL. The column was washed with equilibration buffer (10 CV) at a flow rate of 120 mL/minute. The bound MGG was eluted with 3 CV of elution buffer at a flow rate of 60 mL/minute. The pH of the eluate was neutralized by aseptic introduction of the neutralization buffer. The column was regenerated with 10 CV of regeneration buffer and re-equilibrated with 10 CV of equilibration buffer. Regeneration and re-equilibration were both done at a flow rate of 120 mL/minute. These steps were repeated eight times to process the entire 6-L batch. The total volume used for neutralization of 6 L of eluate was 15 mL.
The purified MGG collected in the eluate bag was pumped into the TFF assembly. The polyclonal MGG was concentrated 16X and constant volume diafiltered with PBS 0.01 M, NaCl 1.5 M, pH 7.4.
After concentration and diafiltration, the purified MGG was pumped across a sterile filter (Opticap phillic filter with Durapore, 0.22-μm membrane) into a bag. A sample was drawn from the final purified MGG to determine the concentration and assess the bioburden of the purified MGG. The purity of the polyclonal MGG was evaluated by running polyacrylamide gel electrophoresis, followed by estimation of purity using QuantiScan software (Figure 2). The bioburden of the purified MGG was determined by plating a sample on growth media and checking for microbial growth. The yield was calculated based on 3% scale results.
Figure 2 shows the purification profile of the polyclonal MGG from mouse serum using the integrated disposable assembly. Comparison of the integrated assembly purified MGG (well 10) to the control reveals that the purity in well 10 was very high. One band each of the light chain and the heavy chain of the MGG were observed and contaminant bands seen in the control were absent. Purity percentages were determined using QuantiScan Software.
The proposed change from concentration/dialysis to ultrafiltration/diafiltration resulted in a significantly faster process and reduced process time from two weeks to 25 hours thus improving turnaround times and productivity at the facility.
The changes to chromatographic buffer composition for both the equilibration buffer (from 10 mM PBS, pH 7.2, to 20 mM PBS, pH 7.4) and elution buffer (from glycine HCL, 0.2 M, pH 2.8, to glycine HCL, 0.1 M, pH 2.8) had the following benefits: an increase in final concentration to 79.28 mg/mL (minimum expected target concentration 25–50 mg/mL); 100% purity (minimum expected target 95%); and an increase in yield of 96.64% (minimum expected target 90%). Reducing filter hold up losses by adopting system rinse and air blow down may increase yields further.
The elimination of the azide also has potential for refinement caused by contamination issues with the dilution buffer. Future work needs to be carried out in order to resolve the contamination issue in the dilution buffer. Using the purification process described, the overall cost of operation for a 200-L batch was estimated to be $41.13/g of MGG.
Gazala Khan-Koticha, PhD, is a consultant for business and knowledge development, Lisa Nakakihara, PhD, is a senior manager for manufacturing operations, Lee Tucker is a scientific manager, John Scarcella is a research scientist, and Eric Rudolph, PhD, is a director for strategy and corporate development, all at Millipore Corporation, Billerica, MA, 781.533.2233, firstname.lastname@example.org