Abstract
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.
Introduction
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.
Original Method
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%.
