Covalent attachment of polysaccharide chains to carrier proteins can produce vaccines with improved immunogenic properties.
These vaccines are effective in infants because they induce isotype switching, thus producing high avidity antibodies and
creating immunological memory. Such glycoconjugate vaccines are available against Haemophilus influenzae type b (Hib), four meningococcal serogroups, and up to 13 pneumococcal serotypes, with a variety of similar vaccines in development.4 Three basic structural classes of vaccines, referred to as neoglycoconjugates, cross-linked matrices, or vesicle vaccines,
can be generated depending on the saccharide hapten (a high molecular weight polysaccharide, or a derived oligosaccharide),
the conjugation chemistry, and the nature of the carrier protein (Figure 2).
Figure 2. Cartoon representations of two classes of glycoconjugate vaccine, showing (a) crosslinked matrix vaccines, and (b)
neoglycoconjugate vaccines. A third class (not shown), glycoconjugates based on outer membrane vesicles, is less common because
we have not be able to obtain NMR data from these samples.
We have obtained NMR (and other) data on the first two classes, with the most data available on the neoglycoconjugates. A
vaccine using CRM197 as the carrier protein typically contains an average of six glycan chains, each of average molecular
weight ca. 5,000 Da attached to amino groups (the N-terminus and the 39 ε-amino groups of lysine residues) in a nonrandom
pattern, so the final conjugate is ~30% carbohydrate. Unfortunately, most carrier proteins are too large or heterogeneous
for detailed NMR analysis (e.g., CRM197 at 58 kDa, tetanus at 250 kDa, and diphtheria toxoids at ca. 59 kDa).
Figure 3 shows the 500 MHz 1 H spectrum of a Hib glycoconjugate vaccine obtained at 30 °C. Resonances from the saccharide chains are sharp and at an identical
chemical shift as those in the native polysaccharide. Conversely, resonances from the carrier protein are broad and ill defined.
This suggests a model in which the carrier protein remains folded, a conclusion supported by circular dichroism (CD) data
of CRM197 before and after conjugation to a synthetic hapten related to the pneumococcal Type 14 CPS.5 In this case, the CD spectra of the free carrier protein and the final conjugate were visibly indistinguishable. The glycan
chains, on the other hand, remain extremely flexible and the conformational space is unaffected by conjugation. NMR analysis
of meningococcal Group C vaccines produces similar conclusions.
Figure 3. Partial 500 MHz 1D 1H spectrum of a Hib-CRM197 conjugate vaccine obtained at 30 °C. The sharp resonances arise from
the covalently attached glycan chains, which retain a very high degree of internal flexibility and have the same chemical
shifts found in the native purified polysaccharide, while resonances from the carrier protein are broad and low intensity,
reflecting the rapid relaxation of the native folded carrier protein. The inset shows a portion of the spectrum of a sample
that has been deliberately degraded, highlighting peaks diagnostic of this.
An excellent method for quantifying the protein:polysaccharide ratio is the deliberate denaturation of the folded carrier
protein by the addition of (deuterated) guanidinium hydrochloride or sodium deuteroxide. The formation of a flexible random
coil results in sharpened protein resonances and the loss of sequence-specific variation in the chemical shifts of peptide
resonances so protons in chemically identical locations resonate at the same frequency. The combination of these factors,
and concomitant de-O-acetylation of the polysaccharide chain when base denaturation is used, allow the polysaccharide:protein ratio to be directly
determined by integration of resonances from the glycan and carrier protein without recourse to methods of poor precision
to independently quantify the saccharide and protein moieties (Figure 4).
Figure 4. Partial 500 MHz 1D 1H NMR spectrum at 70 °C of a pneumococcal conjugate vaccine, dissolved in deuterated water containing 5 M deuterium-exchanged
guanidinium hydrochloride. Denaturation of the carrier protein and destruction of the secondary structure results in a more
flexible random coil structure and loss of sequence-specific chemical shift variability. Resonances from the carrier protein,
such as those from the sidechains of the aromatic amino acids and from the glycan chain, can be integrated and used to determine
the polysaccharide-protein ratio directly.
Proteins Higher Order Structure Information by NMR
The structure of recombinant protein therapeutics is a critical quality attribute because it is directly related to efficacy.
The word structure for protein drug substances such as cytokines and hormones includes three to four elements: the primary structure defined
by the amino acid sequence; the secondary structure elements defined by helices, strands, loops and turns; the tertiary structure
resulting from the assemblage of secondary structure elements; and in multi-subunit proteins, the quaternary structure defined
by the relative positions of the polypeptides with respect to each other.
Currently, various physico-chemical methods (CD, FTIR, MS, fluorescence, peptide mapping) and biological assays provide various
types of information such as the overall folding, the chemical integrity of the polypeptide, and its bioactivity. However,
none of them can provide high-resolution assessments of the structure. Small conformational variations or mutations may be
missed. Furthermore, bioassays cannot detect structural changes that have little or no effect on bioactivity or that may elicit
serious adverse reactions in patients.