INTERNSHIP TRAINING REPORT-2019

TITLE : AFFINITY CHROMATOGRAPHY - an overview

DEPARTMENT : Biosimilars Production - Downstream Processing

BIOCON BIOLOGICS INDIA LIMITED

REPORTING MANAGER : Mr. Prakash. V

SUBMITTED BY : Robin Richard. I

INTERN CODE : P002558

Introduction:

Chromatography is a laboratory technique for the separation of a mixture. The mixture is

dissolved in a fluid called the mobile phase, which carries it through a structure holding another
material called the stationary phase. The various constituents of the mixture travel at different
speeds, causing them to separate. The separation is based on differential partitioning between the
mobile and stationary phases. Subtle differences in a compound's partition coefficient result in
differential retention on the stationary phase and thus affect the separation.

Affinity Chromatography:

Affinity chromatography separates proteins on the basis of a reversible interaction between a

protein (or group of proteins) and a specific ligand coupled to a chromatography matrix. The
technique is ideal for a capture or intermediate step in a purification protocol and can be used
whenever a suitable ligand is available for the protein(s) of interest. With high selectivity,
hence high resolution, and high capacity for the protein(s) of interest, purification levels
in the order of several thousand-fold with high recovery of active material are achievable.
Target protein(s) is collected in a purified, concentrated form. For an even higher degree of
purity, or when there is no suitable ligand for affinity purification, an efficient multi-step process
must be developed using the purification strategy of Capture,
Intermediate Purification and Polishing (Cipp). When applying this strategy affinity
chromatography offers an ideal capture or intermediate step in any purification protocol and
can be used whenever a suitable ligand is available for the protein of interest.

BioProcess Media for large-scale production

Specific BioProcess™ Media have been designed for each chromatographic stage in a process
from Capture to Polishing. Large capacity production integrated with clear ordering and delivery
routines ensure that BioProcess Media are available in the right quantity, at the right place, at the
right time. GE Healthcare can assure future supplies of BioProcess Media, making them a safe
investment for long-term production. The media are produced following validated methods and
tested under strict control to fulfil high performance specifications. A certificate of analysis is
available with each order.Regulatory Support Files contain details of performance, stability,
extractable compounds and analytical methods. The essential information in these files gives an
invaluable starting point for process validation, as well as providing support for submissions to
regulatory authorities. Using BioProcess Media for every stage results in an easily validated
process. High flow rate, high capacity and high recovery contribute to the overall economy of an
industrial process.
Binding: buffer conditions are optimized to ensure that the target molecules interact
effectively with the ligand and are retained by the affinity medium as all other molecules
wash through the column.
Elution: Buffer conditions are changed to reverse (weaken) the interaction between the target
molecules and the ligand so that the target molecules can be eluted from the column.
Wash: Buffer conditions that wash unbound substances from the column without eluting the
target molecules or that re-equilibrate the column back to the starting conditions
(in most cases the binding buffer is used as a wash buffer).
Ligand coupling: Covalent attachment of a ligand to a suitable pre-activated matrix to create
an affinity medium.
Pre-activated matrices: Matrices which have been chemically modified to facilitate the
coupling of specific types of ligand.