Development of a Modified Purification Process for a Monoclonal Antibody Therapeutic-Yield

Case Study

Development of a Modified Purification Process for a Monoclonal Antibody Therapeutic-Yield Improvement

Benefits of the Case:

·  Improved yield of Mab product from bulk harvest by 1.8 fold

·  Simplified downstream purification process by eliminating the collection of fractions and the need for stat assays during manufacturing operations

·  Eliminated the use of resins that were difficult to pack and resulted in declining yields with short life cycles

Background of Challenge:

·  The Mab product had been manufactured at the 2,000L scale.

·  Production at the 10,000L cGMP manufacturing scale was planned.

·  The Mab product harvest contained several product variants.

·  Mab product variants had very similar biophysical properties to Mab of interest.

·  Yield of Mab was unacceptably low.

·  Collection of column fractions was required to obtain desired Mab purity.

·  Host cell modifications were not acceptable.

Strategy

·  Evaluate alternative chromatography resins for efficient removal of the product variants.

·  Eliminate collection of fractions by using step gradients.

·  Optimize process to increase product through put and reduce process costs.

Development Data

·  The purification process was modified by developing an intermediate and final high resolution polishing step.

·  Purity specifications the Mab product were maintained.

·  Yield of Mab was increased 1.8 fold.

Value for the Client

·  The yield for a Mab antibody therapeutic was significantly improved.

·  The purity specifications for the Mab product were maintained.

·  The need for collection and stat analysis of chromatography fractions was eliminated.

·  A significant manufacturing issue was eliminated.

Case Study

Development of a Drop-In Purification Step for Removal of Aggregates from an Antibody Therapeutic

Benefits of Case:

·  Reduced aggregate level in large scale manufacturing downstream process from very high to undetectable levels

·  Simplified downstream purification process by eliminating the collection of fractions and the need for stat assays during manufacturing operations

·  Improved robustness of downstream purification process

Background and Challenge:

·  The client was engaged in large scale cGMP manufacturing operations at a major CMO

·  The client’s product contained high levels of aggregate following an affinity capture step. Subsequent chromatography steps reduced the aggregate level to acceptable levels but product losses were encountered in elution buffers, and stat in-process assays. The process was not robust. CMO did not want continue manufacturing product under current conditions.

·  The client desired to improve the purification process efficiency by introducing either an aggregate disassociation step followed by purification or an efficient protein aggregate removal step compatible with large scale biopharmaceutical manufacturing.

Strategy:

·  Evaluate chemical disassociation of the aggregate followed by product purification

·  Evaluate chromatography resins for efficient removal of the aggregate

Development Data:

·  Conditions were established to disassociate the product aggregate with chemical treatment but the biological activity f antibody was reduced

·  A single drop-in chromatography step was developed that reduced aggregate levels to undetectable levels with essentially no product loss

·  The new process step was compatible with the existing purification process; i.e. no modifications to existing process were required

·  Limits for key process parameters were established

·  New chromatographic process step was successfully scaled and transferred to major CMO

Value for the Client:

·  Manufacturing process for antibody therapeutic was significantly improved and streamlined

·  Aggregate levels were reduced to undetectable levels

·  The need for collection and stat analysis of chromatography fractions was eliminated

·  New process step eliminated downstream process bottleneck

·  Significant strategic business alliance was facilitated with elimination of manufacturing issues

Case Study

CMO’s Cannot Produce SP2/0 Working Cell Bank: Cytovance Identifies Problem and Successfully Produces WCB

Benefits of the Case:

·  A viable Working Cell Bank (WCB) that thaws with good viability and demonstrated logarithmic growth necessary for expansion

·  Further
Characterization of Cell Line

Background and Challenge:

·  The client had purchased a Master Cell Bank that had been manufacturing in 1999.

·  The client is able to grow cells successfully in the R&D laboratory.

·  The client transferred vials of MCB to a well-known CMO to manufacture a WCB

·  The CMO failed several times to successfully manufacture a WCB

·  Poor viability and no growth

Strategy:

·  Cytovance to perform tech transfer utilizing data from Client R&D and CMO

·  Cytovance to manufacture Engineering lot using tech transfer parameters from R&D to demonstrate transfer

·  Engineering Run data:

·  The MCB thawed with a good viability and had adequate growth to scale-up to achieve cell numbers to lay down 350 vial WCB

·  Post Freeze Testing of the engineering run showed good viability at thaw, but viability declined and growth was not achieved.

·  The beginning, middle, and end of bank was tested with the same results

·  Filling process run at room temperature and cells were exposed to DMSO for 60 minutes

·  Cryogenic freeze profile utilized forced nucleation

Development Strategy:

·  Using MCB utilize Manufacturing and Process Development technicians to study DMSO hold times

·  Using MCB utilize Manufacturing and Process Development technicians to study room temperature versus cold process temperatures

·  Using MCB utilize Manufacturing and Process Development technicians to study Cryogenic freeze profile (1°per minute, < 1° per minute)

Value for Client:

·  A GMP run was commenced with the new freeze profile and after Post Freeze Testing of the newly manufactured WBC the bank thawed with good viability and achieved the required growth rate

·  The clients may now continue production and get their product into clinical trials

·  Client ecstatic!

Case Study

Rapid 200 Fold Scale-Up of a Fed-Batch Bioreactor Process for a CHO Cell Line expressing a Mab

Benefits of Case:

·  A fed-batch bioreactor process of a CHO cell line was transferred from a client and scaled 200 fold with minimal process development evaluation and testing

·  Equivalent productivity profiles were obtained at 0.5, 3, and 100L bioreactor scale

·  A commercially available off the shelf chemically defined media and feed solution were utilized

·  Process Development costs and time were minimized

Background and Challenge:

·  The client had performed preliminary bioreactor runs at the 0.5 scale with the CHO cell line

·  The client required a rapid 200 fold scale-up to the 100L bioreactor level with minimal process development

·  Time constraints dictated that only two-3L bioreactor evaluations could be performed prior to scale-up to the 100L bioreactor

·  The clients’ 0.5L bioreactor system differed in design from the 3 and 100L bioreactor utilized for process development and scale-up

Strategy:

·  Utilize a commercially available off the shelf chemically defined media and feed solution used by the client at the 0.5L scale

·  Utilize process data from the 0.5L sale to design operating parameters for the 3L scale bioreactor although the 0.5 and 3L bioreactors differed in design

·  3L process development and 100L scale bioreactor are of similar design and use the same control software

Development Data:

·  The most favorable integrated viable cell density was obtained with the 0.5L bioreactor most likely due to oxygen delivery rates

·  A productivity of 900mg of IgG/L at the 0.5 scale transferred to the 3 and 100L bioreactor scale was achieved

·  The feed schedule data from the 0.5L scale transferred to the 3 and 100L scale with only minor adjustments

·  Metabolic profiles were similar at all three bioreactor scales

·  Only two process development 3L bioreactor runs were required prior to scale-up of the process of the 100L bioreactor

Value for the Client:

·  A rapid 200 fold bioreactor scale-up was achieved with minimal process development

·  Use of a commercially available off the shelf chemically defined media and feed solution facilitated technology transfer and scale-up

·  Process development costs were minimalized and product launch accelerated