Biomedical Engineering Reference
In-Depth Information
X
Severity
Figure
4.19.
Criticality of glycosylation. (See the insert for color representation of this figure.)
noncritical quality attribute. The major oligosaccharide forms are defined as produc-
t-related substances, and therefore routine monitoring is not required.
4.2.2 Case Study 2: Deamidated Isoforms
Prior Product Knowledge
L
ABORATORY
S
TUDIES
. Deamidation at Asn or Gln residues is a common occurrence
in human proteins [30, 31] and recombinant monoclonal antibodies [32]. Asn-Gly
sequences are present and conserved in the constant regions of IgG molecules, and these
sites are known to undergo deamidation under physiological conditions. The resulting
charge isoform heterogeneity can be readily detected by using native IEF, which shows
the formation of more acidic bands. The charge isoforms were further characterized
by fractionating motavizumab using ion-exchange chromatography (IEC) as shown Fig.
4.20.
Peptide mapping of IEC fraction 1 (more acidic isoforms) and IEC fraction 2 with
online mass spectrometry (MS) demonstrated that the major deamidation sites are
located in the Fc region. The primary deamidation site is Asn-387 on the heavy chain as
seen for other antibodies [33, 34]. Other identified deamidation sites inmotavizumab that
were detected at lower levels are Asn-157, Asn-289, Asn-318, Asn-328, Asn-424, and
Asn-437. Because the bioactivity is independent of Fc function, deamidation is unlikely
to have an effect on the activity of the molecule. As expected, the deamidated isoforms
(IEC fraction 1) exhibited similar F protein binding activity to motavizumab and IEC
fraction 2, as measured by ELISA and SPR, and were able to neutralize RSV (Table 4.4).
Motavizumab was also incubated in human plasma at 37
C for up to 5 weeks and
analyzed by both IEC and native IEF. Native IEF analysis demonstrated that acidic bands
were generated during incubation in human plasma at 37
C (Fig. 4.21). IEC analysis also
showed an increase in acidic isoforms with time.
To demonstrate that the acidic isoforms observed by IEC and native IEF were due to
deamidation, peptide mapping was performed on all samples recovered from human
