CCAB Blog – Antibody production: a week in the life

By CCAB - September 16, 2019 10:02 am (leave your thoughts)

How does a CCAB scientific associate spend her week? Let’s find out.

By Kaajal Nagar
Scientific Associate

I’ve always loved and been curious about nature but my interest in science really began during high school and developed during my undergraduate degree. Once I started my MSc, I realized that scientific research is actually very slow and often frustrating because things rarely work how you plan and usually don’t work how you want them to. Being a scientist has taught me how to: be patient, persevere, problem solve and think creatively.

So what was my path to my current job? I completed my BSc in Biochemistry from the University of Guelph and an MSc in Biochemistry from the University of Toronto. Before joining the Centre for the Commercialization of Antibodies and Biologics (CCAB), I worked in academia, at contract and large pharmaceutical companies and with small biotech companies. I’ve been a scientific associate at CCAB for three years. For the first two years, my workflow focused largely on IgG production to support commercialization of the University of Toronto’s antibody assets. Last year our company launched C-Lab, a products, services and solutions division of CCAB, which provides a client-focused contract research organization experience for both academia and industry.

My day-to-day activities at CCAB involve designing and performing experiments, documentation and analysis of the results. I also write scientific reports and protocols, present my results, perform literature searches and critically evaluate other’s scientific findings. I am the lab representative on the Biosafety committee and spend time actively ensuring the health and safety of my lab colleagues. As with any lab, I am responsible for a number of weekly chores that ensure the lab is well organized, cleaned and stocked with the reagents we need to conduct our experiments.

A week in the life of producing IgGs

At C-Lab, our expertise is in recombinant antibody production and, to date, we have successfully expressed and purified many antibody species, isoforms and formats. The majority of our production work is done with the IgG molecule. The nice part about making antibodies is that the C-Lab team has the tools, knowledge and experience to ensure that these experiments almost always work. The team’s expertise allows us to meet the varied needs of our clients. Some are researchers that require an antibody for a specific target and only need small amounts of an IgG. Other clients need a larger amount of IgG for later stages such as in vivo studies. The team performs small scale (2 mL), medium scale (10 mL, 40 mL, 200 mL) and large scale (litres) of IgG production depending on the amount that is required.

My typical week of IgG production involves doing several 40 mL or 200 mL purifications.

Wednesday – Seeding cells for transfection

IgG production begins by setting up flasks of cells for transfection. We grow suspension cells that have been specifically engineered to produce large amounts of IgG. These cells are grown on shaking platforms in our incubators. It is very important that these cells are well maintained, healthy and are seeded at the correct density.

Thursday – Transfection day

On transfection day, I begin by warming my transfection media. Transfection is the process of introducing DNA into cultured cells. I calculate the amount of DNA I will need to add to each transfection and check the DNA preps that have been made for me. The DNA constructs that I transfect are in a vector that has been engineered to maximize IgG expression. These constructs allow for the expression of heavy chain (HC) and light chain (LC).

Prior to beginning my transfection, I check the cells to ensure that they are at the correct density for transfection and have high viability. For each flask, I co-transfect a plasmid that allows for the expression of HC DNA and a plasmid that allows for the expression of LC DNA. The cells are then placed back in the incubator for 4 days in order to allow for the expression of IgG. At this stage, it is important to maintain sterility of our production line, therefore this work is done inside a room specific for tissue culture work with specialized workstations called biosafety cabinets (BSCs). When there is a lot of activity going on in the lab, I find it peaceful to work in the tissue culture room by myself for awhile. The hum of the BSC is calming, almost meditative, and doing a transfection is a short period of focused work. This is my favourite part of purification week.

Friday to Monday – IgG is produced by the cells

The cells that have been transfected express IgG and this IgG is then secreted into the conditioned media. During these days, I work on other client projects, updating my notebook, documentation and I complete the work required for the previous week’s purifications.

Tuesday – Harvest day

For each transfected flask, I count the cells and record the number of cells and viability. This information is useful because the expression of some IgGs is toxic to cells and may result in low production yields. I then centrifuge the transfected cells and collect samples of the pellets and conditioned media. The remainder of the conditioned media is then incubated with Protein A beads overnight on rotors in the cold room to allow for maximum binding of IgG to the beads.

Wednesday – Purification day

This is the busiest day of the week in the lab (and my supervisor’s favourite day!). Each conditioned media and bead mixture is poured into a column and the flow through drips out of the column. This process can take an hour or two depending on the amount of IgG and beads in the sample. This is the most frustrating part of the purification and requires the most patience. I’m pretty sure that the more you watch the columns, the slower the liquid drips out. The beads in the column are then washed multiple times with DPBS. For the final step, the bound IgG is released from the beads using an acidic elution buffer and the eluted IgG is collected. This eluted IgG is then neutralized and concentrations of the eluted IgG and neutralized IgG are measured in order to determine initial yields.

Throughout the purification process, samples of the flow through, wash and eluted IgG are collected. These samples and the pellet and conditioned media are analysed by SDS PAGE. The gels help us determine at which step there is a problem if there are issues with the purification.

The IgGs are then concentrated and buffer exchanged into 1XDPS using centrifugal filters. For larger purifications (200mL), the IgG is dialysed overnight and the following morning into 1XDPBS.

Thursday – Completion of IgG purifications and beginning of quality control

I complete buffer exchanging and concentrating the IgGs. The IgGs are then sterile filtered, final concentrations are measured, IgGs are labelled, and the final yields and details of each purification are reported.

As a team, we then complete quality control. Each IgG is run on SDS-PAGE in reducing and non-reducing conditions to ensure that it is the correct size and high purity. We also perform endotoxin tests to show that the produced IgG is clean and can be used on cells or in vivo.

Friday – Completion of a typical purification week

At the end of the week, any remaining quality control tests are completed. All documentation is completed, client forms are filled out and datasheets for each IgG are created. Also, the IgGs are aliquoted into 2 mL tubes, labelled and prepared for shipment to the client.

The part I like most about my job is the satisfaction that comes from eventually figuring things out, working with others on a common goal and knowing that I am spending my time and effort producing antibodies that could be valuable tools for research or could be potentially developed into therapies to treat cancer and other diseases.

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