CATION from the sample. The samples were

 

CATION EXCHANGE
CHROMATOGRAPHY

Charge heterogeneity was assessed using cation exchange
chromatography (CEX). Many  presumptive
post-translational modifications may lead to variations of monoclonal
antibodies charge distribution that can conceivably affect their activity and
process consistency. The characterization of these charge variants serve as a
crucial quality control step to ensure stability and efficacy. mAbs charge
variants can be assessed by carboxypeptidase digestion using cation exchange
chromatography.

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 Ion exchange chromatography is a technique
based on the electrostatic interactions between the resin functional groups and
the analyte. This method is used to determine the acidic, basic, and main peak
fractions from the sample. The samples were diluted to 1.0 mg/ml with milli Q
water and were either treated with carboxypeptidase enzyme for C-terminal
lysine digestion and incubated at 37°C for 2 hours or analysed as intact sample.
After digestion, the samples were separated on a weak cation exchange column (4.0 mm x
25 cm length, 10 µm particle size). Gradient elution was used to optimize
elution conditions using phosphate buffers with properly titrated pH. Detection was carried out at 214 nm. After
the protein of interest has been eluted, proteins that remain bound to the
column resin are eluted by increasing the ionic strength of the elution buffer. When analysed, the
peaks which elute earlier than the main peak are known as acidic species while
basic species are the variants that elute later than the main peak.

SIZE EXCLUSION
CHROMATOGRAPHY

Size-exclusion
chromatography (SEC) is a chromatographic method used for the detection,
characterization and precise quantification of protein aggregates in biological
products Separation of different
compounds according to their size is measured by size exclusion chromatography.
 The quantitation of aggregates is a matter of
concern given their plausible effect on efficacy and immunogenicity . Separation occurs when molecules of
different sizes are passed through the column packed with resins.  Large molecules are
completely diffused from the pores and pass through the space in between the
gel matrix and elutes first while smaller molecules gets allocated in between
the mobile phase of in and outside the molecular sieve and their flow through
the column is retarded, hence appear later in effluent . Therefore, molecules separate based on
their size and elutes in order of their decreasing molecular weight. Samples
are eluted isocratically from a SEC column, using a single buffer system. The
samples under examination were diluted to 10mg/ml in milliQ water. The buffer
conditions were selected that are compatible to the protein stability and activity.
0.2M dibasic sodium phosphate buffer was used and the pH of the mobile phase
was adjusted at 7.0±0.1, with 0.2M monobasic sodium phosphate. After sample
application the entire separation takes place as one column volume of buffer
passes through the colum. The samples were run
at a flow rate of 0.5ml/min to obtain better resolution. For SEC analysis UV
detection at 280nm was used for greater response.

The peaks eluting
before main peak are known as aggregates while the later eluting peaks are
known as fragments.

CAPILLARY
ZONE ELECTROPHORESIS

The
capillary zone electrophoresis (CZE) technique is one of the most sensitive and
high resolution separation assays for the analysis of biomolecules..  An expeditious and
high-resolution capillary zone electrophoresis (CZE) method capable of
resolving the charge isoforms of intact Rituximab has been used to monitor the
charge heterogeneity .Separation was carried out using
a neutral capillary with a buffer system composed of  50 mM -amino-n-caproic acid containing 0.2
per cent HPMC, adjusted to pH 4.50 with glacial acetic acid. The effective
length and total length of the capillary was 40cm and 50 cm respectively. The
sample is diluted to 0.5mg/ml with milli Q water .Applied potentials as high as 30
KVolts were used to drive the electrophoretic process. Such high potentials
promote rapid migration of zones, while minimizing zone spreading. The capillary
temperature was 20±2 °C. The detection wave length was 214 nm with
injection time of 1psi for 10 sec.

CAPILLARY
ELECTROPHORESIS- SDS PAGE

Capillary
electrophoresis dodecyl sulfate (CE-SDS) overtures a rapid, sensitive and perceptible
method for the analysis of biomolecules. Both reducing and non-reducing CE SDS gel conditions were applied and
optimized to characterize therapeutic monoclonal antibody. For the
non-reduced analysis, the native protein was treated with SDS prior to
separation to mask the protein native charges. For reduced analysis, the sample
was treated with SDS, and beta-mercaptoethanol (BME) to reduce the native
protein structure. All the antibody samples were diluted to obtain a minimum
concentration; within the range of 0.2 mg/ml to 2 mg/ml according to the
following procedure:  68.0 µL HPLC grade
water was combined with 100 µL SDS sample buffer, 2 µL of 10 kDa internal standard,
10 µL mercaptoethanol and 20 µL test solution in a 1.5 mL eppendorf tubes. The
solution was mixed, centrifuged at 3000rpm for 1min and heated at 70° C in a
water bath for 10 minutes. The mixture was cooled at room temperature for at
least 3min and transferred to sample vials for injection. For
non-reduced analysis, 250mM iodoacetamide (IAM) during heat treatment
of the non-reduced sample, combined with optimization of incubation temperature
and time (70ºC for 5 minutes, in the presence of SDS), was
able to significantly suppress the extent of thermally-induced fragmentation. A
PDA detector at 220nm was used in conjunction with 30.2 cm × 50 ?m; Bare fused
silica coated capillary. The capillary temperature was maintained at 25 ± 2°C. Under
reducing condition, the percentage of non-glycosylated heavy chain (NGHC) in
the solution was calculated whereas under non-reducing condition the percentage
of impurities in the sample solution was calculated.

Therapeutic
monoclonal antibodies , will be reduced to glycosylated heavy chain (HC), non-glycosylated
heavy chain (NGHC) and light chain (LC).

Capillary Isoelectric Focusing
(CIEF)

 Iso electric focussing is based on the fact
that each protein has a different pH at which it is electrically neutral; its
isoelectric point (pI). Thus, each sample component migrates to its own
isoelectric point and gets separated by electrophoresis in a pH gradient
generated between the cathode and anode. A
two-step process was performed: first, in which proteins were focused in the
capillary and second, displacement of the zones out of the capillary by means
of changing the chemical composition of anolyte or catholyte solution. In this
method, the change in anolyte or catholyte causes a shift in the pH gradient,
resulting in migration of the zones past the detection point in the capillary.
The method involves the use of coated capillaries to prevent undesirable
adsorption of analytes onto the capillary inner surface. The process involves
addition of 200 µL 4.3 M Urea, 12.0 µL of Pharmalyte 3-10 carrier ampholytes,
20.0 µL of 500 mM Arginine, 2.0 µL of 200 mM Iminodiacetic Acid, 2.0 µL of pI
marker 10.0,pI marker 7.0,pI marker 5.5 and pI marker 4.1 and 10 µL of  sample with a protein concentration within 5
mg/ml to 10 mg/ml and salt concentration below 50 mM ,since high salt content
is detrimental to cIEF separation. The master mix is vortexed for 15 seconds to
ensure complete mixing. 242 µl of the master mix is added to all the labelled
sample tubes as well as the pI marker tube. To all the sample tubes 10µl of
sample (diluted to 1 mg/ml with sterile DDI water) is added with 2 µl of the
remaining pI marker to the pI marker tube. 
The master mix with protein is vortexed for 30 seconds and 180 µl of the
sample is transferred into the micro vial placed inside the universal plastic
vial taking care not to introduce bubbles at the bottom for further analysis. A
plug of sample is introduced into a Neutral Capillary, 50 ?m internal diameter
and length about 40 cm and detected at a wavelength of 280nm.