Bristol-Myers Squibb (BMS) – Rensselaer Polytechnic Institute (RPI) – Cooperative Education Work Report

Rensselaer Polytechnic Institute i 12/3/2013

Cooperative Education Project Work Report Rensselaer Polytechnic Institute

Evaluation of Protein-A Wash Conditions for Antibody Purification

Duration: 13JAN14 – 03JUL14

Ray Parker1

Rensselaer Polytechnic Institute, Bachelor of Science, Chemical Engineering

Bristol-Myers Squibb, Hopkinton, Massachusetts 01748

Mentors: Srinivas Chollangi Ph.D.2, Yi Li Ph.D.3

1 Downstream Process Development Co-op, Chemical Engineering, Rensselaer Polytechnic Institute. 2 Scientist I, Chemical Engineering, Bristol-Myers Squibb, University of Oklahoma – Norman, University of Texas. 3 Senior Scientist, Chemical Engineering, University of Delaware – Newark, University of Arkansas

Bristol-Myers Squibb (BMS) – Rensselaer Polytechnic Institute (RPI) – Cooperative Education Work Report

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I. Part One Bristol-Myers Squibb is a specialized biopharmaceutical company that focuses on the discovery,

commercialization process development, clinical trials and commercialization of new drugs. Bristol-Myers Squibb

manufactures prescription pharmaceuticals in several therapeutic areas, such as cancer, HIV/AIDS, cardiovascular

disease, diabetes, hepatitis, rheumatoid arthritis and psychiatric disorders. Bristol-Myers Squibb’s mission is to

discover, develop and deliver innovative medicines that help patients prevail over serious diseases.

The entire pipeline process starts in the Cell Line Development, where each specific cell line that codes for the

specific antibody is developed. Then the antibody must be purified and a process must be developed in order to purify

the antibody to be injected into patients. Development of the commercial purification process falls to the responsibility

of the Process Development Department. Once a purification protocol is finalized, the drug substance must undergo

testing in the Clinical Trials Phase. Once extensive studies have been completed on the antibody substance, the drug

can then move onto commercialization development to be manufactured and then sold to patients around the world.

Co-op Abstract. Under the direction of BMS mentor, the Co-Op will work with the

Downstream Process Development team to develop novel separation technology and gain further

understanding of the current protein purification platform. The project will focus on key unit operations

such as ion-exchange, hydrophobic, and mixed mode chromatography to improve impurity clearance

and product yield. Experimental work will include process condition optimization, process

characterization, as well as process understanding at the molecular level. The Co-Op will collaborate

with BMS TECAN team (from multiple sites) to implement high throughput screening tools and will

use design of experiments (DOE) methodology to map out operational space. In addition, biophysical

characterization of glycoprotein structure will be performed to assist process understanding.

A. Key Deliverables at Project Completion:

1. Develop chromatography conditions to separate charge variants, aggregate populations, and

different glycol-forms.

2. Characterize heterogeneous protein populations and impurities (when applicable) with various

biophysical and biochemistry tools.

Cell Line Development Harvest Development Purification Process Final Drug Substance

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3. Study mechanistic and empirical correlations between process parameters and product attributes

and provide insight to current process issues (such as impurity clearance and product stability).

B. Key Learning:

1. Protein bio-separation techniques in an industrial setting.

2. Process analytical and characterization tools.

3. Downstream process development essential knowledge.

Process Development or PD, focuses on the developing methods for expressing and purifying the drug substance

to move onto formulation. PD consists of two sub-departments; Process Development Upstream and Process

Development Downstream. Process Development Upstream uses the cell line developed by the Cell Line

Development Department to optimize host cell growth and antibody/titer production to produce harvest, which is a

mixture of cell debris and antibodies. The harvest is then processed in Downstream Process Development, which

removes host cell proteins and other impurities.

Downstream Process Development helps to ensure that the quality of the drug remains consistent and robust. The

process in Downstream Process Development goes from Clarification (Centrifugation/Depth Filtration), to Capture

Chromatography (Protein-A/Viral Inactivation) to Intermediate Purification Chromatography to Polishing

Chromatography then to Viral Filtration. For each of the chromatography steps, there are sanitization, equilibration

and load steps. Then following the load step, there are a various number of wash steps, which are then followed by

elution, strip/regeneration and then sanitization, neutralization and storage of the column. During the eluate phase, the

elution is collected and processed further down the pipeline.

Strategies for separating monoclonal antibody are changing from molecule specific procedures to platform

strategies. With the help of high-throughput testing strategies platform process has become increasingly robust and

effective, removing more process and product related impurities such as DNA, HCP, CHO Cells & High Molecular

Weight (HMW) and Low Molecular Weight (LMW), respectively. The Capture Step (Protein-A), or affinity

chromatography, is the first step in downstream process development, which involves binding to the target antibody.

By effectively optimizing Protein-A, we can reduce the workload on the other columns downstream of the Capture

Step, helping to make the process more robust and efficient, which can reduce the amount of chromatography steps

required, saving time and money for BMS.

In order to find out which buffer/resin/molecule combinations are most effective in removing non-specific binding

interactions between HCP’s and mAb’s, screening tools were used.

High-Throughput Screening Optimization • For each chromatography condition, buffer conditions were made with a multi-channel pipette in a separate

96 well plate before testing. Harvest material was then filtered and prepared for Protein-A chromatography.

The PreDictor resin plates were equilibrated with equilibration and sanitization buffer. The protein, Anti-

Lag3, was then loaded onto the resin. The resin was washed with equilibration buffer, the variable excipients

wash, and another wash step to remove any unbound material. The column was eluted and collected and

then stripped with a harsher buffer. The column was cleaned with strip buffer and stored in alcohol. Tests

were repeated with different buffers that had different pH values of 5.5, 7.0 and 9.0.

Column Screening Optimization • The Wash pH Study consists of a similar protocol as the Wash Excipients Study, but rather than preparing

different excipients washes, different wash buffers at pH values ranging from 3.5 to 8.5 (in .5 increments)

were used to see the effects of pH on each molecule.

Viral Inactivation Process Optimization • The Viral Inactivation Study deters the optimal pH that removes the most Host Cell Proteins (HCP) and

other aggregates. Protein A elution was titrated down to pH 3.5, held for one hour, then brought back up.

Results were then analyzed for DNA, Titer and HCP.

Analytical Techniques • Chromatograms were run on GE AKTA™ Avants. Turbidity and UV readings were tested in Agilent

Technologies Cary 60 UV-Vis at A280nm & A410nm. Monomer content of the pooled flow through fractions

was analyzed by size exclusion chromatography using Acquity UPLC. ELISA Assays were run using

TECAN Freedom EVO®. Statistical analyses were performed using Microsoft® Excel and SAS JMP 8.0.

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The goal for the duration of this co-op rotation was to identify Wash and Viral Inactivation strategies that improve

impurity clearance of Protein-A capture Chromatography, making the process more robust and efficient. This is

important because the Capture step is one of the most important steps since it is early on in the purification process,

and can potentially reduce the total number of chromatography steps required after Protein-A, which can save money

and time for BMS. With the reduction of impurities, it also reduces the load on the later downstream development

chromatography steps making the process more consistent and effective.

II. Part Two In comparison to the job description and my expectations at BMS, I was pleased. I expected to be able to;

Develop chromatography conditions to separate charge variants, aggregate, and different glycoforms.

Characterize heterogeneous protein populations and impurities with various biophysical/biochemistry tools.

Study mechanistic and empirical correlations between process parameters and product attributes and provide

insight to current process issues (such as impurity clearance and product stability).

Learn about Upstream and Downstream Process

Learn at least one new computer software/application.

Be able to describe the entire process development process.

Be able to present well on material by the end of the co-op

Be a contributor to a publication in while on co-op.

All of my goals have been met, besides being a contributor to a publication while on co-op; this may happen later

on. My mentor, Srinivas Chollangi, has been very patience while working with me on the concepts of Process

Development, which do not come naturally for me. In addition to this, my colleagues that I share laboratory space

with, have been very helpful in my development as a scientist too. They have all given me suggestions on how to do

things differently to improve my skills as well as given me conceptual advice on what could be done more effectively

while running experiments and other concepts in Downstream Process Development.

The main objective of my co-op project was to reduce the amount of chromatography steps required to purify

certain drug substances in order to reduce costs, since chromatography resin very expensive (upwards to $15,000 for

one liter). During the project, I was able to optimize chromatography processes for one capture step so effectively that

we were able to get the same purity with one column and one filter that the older process was able to achieve with

three columns and one filter. When scaling up to a commercial process, this could reduce costs.

In addition to learning about Downstream Process Development, I have had the opportunity to work in other field

as well, such as in Upstream Process Development and the Process Development Analytics Testing Group as well. A

list of skills learned and concepts introduced are listed below. In addition to these skills, I have accomplished much

during my time at BMS, including; making over 400 buffers, running over 100 Avant runs, helping on five different

molecules, running over 25 DBC tests, running over ~6 studies and <60 different experiments, packing over ~50

columns, and helping over 10 colleagues with testing/buffers/sample prep.

Downstream PD Concepts • GE Pilot, Avant, & Explorer Usage

• Column Packing & Testing

• Buffer Making and Preparation

• Protein-A Resins Usage

• AEX/CEX Resins Usage

• HIC Resins Usage

• Membrane Chromatography

• Resin Amount Calculation

• Residence Time Calculations

• High-Throughput Screening Tools

• Amount of Harvest to Load

• DOE Experiments Testing

• Viral Filtration Setup

• Viral Inactivation Process

• Tangential Flow Filtration Setup

Software

• GE UNICORN Software

• Multi-Genetic Algorithms (MGA)

ADT Concepts

• ELISA - Dilution Plates Preparation

• A410 Testing

• A280 Testing

• HPLC/UPLC Exposure

Miscellaneous Concepts

• 6S Orientation Preparation

• GMP/Laboratory Safety Training

• Sample Data Mining

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No patents or honors/rewards were received. Various publications and presentations may represent my work in

the future.

Rensselaer’s Co-op Program has been very insightful and has been a great learning experience. I am thankful for

the opportunity to be able to go on co-op. My only comment to make the co-op program better would be to have the

monthly emails sent out on time at the beginning of the month. I cannot think of anything different.

Overall, my experience at Bristol-Myers Squibb has been very interesting and useful to my future. I have never thought

that I would be able to have a clear understanding of how some pharmaceutical companies work, or the basic process

behind how certain mAb drug substances are made. In addition to gaining an understanding of the process, I’ve been

able to help accomplish very important goals that will have a fairly large impact on the company and my work will

help to reduce costs and save time for BMS when developing drug molecules that I have been working on.