New technology for the recovery and purification of recombinant proteins using a continuous-flow system via voltage-dependent gating in a single step.

The new process replaces the traditional method of chromatography, which requires tonnes of ion-exchange resin and is very time-consuming.
The recovery, isolation and purification of recombinant proteins using traditional methods is complex, time-consuming and therefore expensive. This is particularly true for pharmaceutical products such as monoclonal antibodies (mAb).
The mAK-producing cells (usually CHO cells) and cell debris in the fermentation broth are separated from the liquid by centrifugation. This is followed by depth filtration, which completely removes any remaining small particles and turbidity, resulting in a clear, cell-free supernatant.
The subsequent primary purification (capture) in this step involves capturing and concentrating the antibodies from the protein mixture. The use of Protein A affinity chromatography is the tried-and-tested standard here. The column matrix contains Protein A, which binds specifically to the Fc region of the monoclonal antibodies. Other proteins and media components are simply washed away.
Elution: By lowering the pH (to an acidic level), the antibodies are released from the matrix and collected in a highly pure form. The acidic pH is maintained for some time to inactivate any enveloped viruses that may be present.
Polishing using hydroxyapatite: To achieve a level of purity suitable for pharmaceutical products, contaminants (e.g. host cell proteins, DNA, antibody fragments or aggregates, and viruses) are removed.
This is followed by the cation-exchange purification step (CEX), which separates the charge variants of the mAbs and aggregates. The antibodies pass through the column. This is followed by virus filtration using nanofiltration to remove even non-enveloped viruses. The next step is UF/DF: the antibody solution is concentrated and the buffer is replaced with the final storage medium (formulation buffer, which guarantees shelf life). Fill & Finish involves sterile filtration into the final containers.
However, these established methods have serious technical, economic and environmental drawbacks and are not continuous processes. They exhibit incomplete selectivity and mixed fractions, as the binding affinities on chromatographic matrices partially overlap during salt or pH elution.
The cycles of loading, washing, eluting and regenerating the column drastically limit the continuous mass flow in an industrial production line and require oversized equipment. At high salt loads, the desalination effort is considerable. The use of highly concentrated salt buffer systems for protein elution necessitates downstream desalting steps, e.g. by means of expensive dialysis/diafiltration or ultrafiltration, in order to convert the target proteins into a low-salt form ready for use.
As recombinant proteins are always present as a heterogeneous mixture in the isoelectric range, the proposed method acts as a time-based molecular sieve in millisecond on/off mode. The new system is a prime example of continuous mass separation.
Through the careful selection of the pulse frequency - duty cycle - and the pH gradient, the passage of the protein through the CEM can be precisely controlled. The system utilises IEP for migration-based separation, not for conventional precipitation. The protein therefore remains in solution in its native form.
The process is designed to enable continuous, highly selective and product-friendly separation of recombinant proteins directly within the liquid stream, without the need for adsorptive binding to a solid phase or the use of salt gradients, whilst guaranteeing the highest purity and yields on an industrial flow-through scale.
The core of the innovation lies in the targeted selection of the pulse frequency - specifically the pulse-pause cycle - in order to exploit the diffusion rates of different isoforms at the IEP for separation. Furthermore, it involves precisely controlling the duty cycle and the pH gradient for the passage of the protein through the CEM in the millisecond range as a Ton/Toff signal.
The way in which the system utilises the IEP for selective separation is described as follows. As the system operates with a CEM, only positively charged molecules - that is, cations - can pass through the membrane. The condition in the feed stream is pH < IEP: as long as the recombinant protein is protonated and positively charged, it migrates through the CEM into the second chamber under the influence of the electric field. The system utilises the IEP for migration-based separation, not for the conventional precipitation of proteins.
Lactoperoxidase (LPO) and lactoferrin (Lf) can be separated very effectively using this technology and recovered with the highest purity and yield.
The system has the potential to revolutionise pre-clarification and intermediate purification (capture/intermediate) and operates continuously in a flow-through mode, which is regarded as the gold standard in biotechnology.

Norbert Pautz - Managing Director
Bioway Consulting SL
C./ Tirant Lo Blanc 4 , office 19
ES-03750 Pedreguer / Alicante - Spain
Phone 0034965030786
Mail contact@biowayconsulting.com
www.biowayconsulting.com
Presscontact: Mr. Norbert Pautz

Bioway has been in existence for almost 40 years and was founded as a sole proprietorship in Germany. A limited liability company was established in Switzerland with a subsidiary in Germany and later as a private limited on shares in Cyprus. Now since November 2024 Bioway Consulting SL is incorporated
Bioway deals with electrolysis-based wastewater treatment technologies, incorporating complementary technologies such as membrane filtration. Furthermore, a system for air purification / disinfection was developed to inactivate infectious biological particles such as viruses and bacteria.
Bioway works as a consultant mainly in D, A, CH - German-speaking countries, whereby new technologies are further developed and sold on the market in co-operation with system manufacturers.
The main task is to advise customers, solve problems, implement technology to solve tasks and sell systems / equipment for the purification of liquids (e.g. cooling lubricants) and industrial waste water. R & D take place in an own laboratory is Spain. New project again is protein extraction and purification with electrolytic methods.

This release was published on openPR.