Liquid chromatography of recombinant proteins and protein drugs

Geng, Xindu; Wang, Lili

doi:10.1016/j.jchromb.2008.01.041

Cited by 62 publications

(21 citation statements)

References 158 publications

(184 reference statements)

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“…Hydrophobic interaction chromatography (HIC) has been widely employed in the separation of proteins as it typically separates species based upon molecular hydrophobicity [10][11][12][13][14]. Proteins can be separated rapidly with high resolution at high yield without denaturation by gradient elution with decreasing salt concentration.…”

Section: Introductionmentioning

confidence: 99%

HIC resolution of an IgG1 with an oxidized Trp in a complementarity determining region

Boyd¹,

Kaschak²,

Yan³

2011

Journal of Chromatography B

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Section: Introductionmentioning

confidence: 99%

HIC resolution of an IgG1 with an oxidized Trp in a complementarity determining region

Boyd¹,

Kaschak²,

Yan³

2011

Journal of Chromatography B

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“…In general, recombinant protein purification for vaccine purposes requires high purity and a process able to maintain bioactivity of the protein at the end of the downstream process; thereby, expensive and more complex purification technologies are required, which lead to increased costs of the process (Geng and Wang 2008). Liquid chromatography is a largely well-established tool in industrial scale purification (Noble 2001).…”

Section: Introductionmentioning

confidence: 99%

Development of production and purification processes of recombinant fragment of pneumococcal surface protein A in Escherichia coli using different carbon sources and chromatography sequences

Carvalho

Cabrera-Crespo

Tanizaki

et al. 2011

Appl Microbiol Biotechnol

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Pneumococcal surface protein A (PspA) is essential for Streptococcus pneumoniae virulence and its use either as a novel pneumococcal vaccine or as carrier in a conjugate vaccine would improve the protection and the coverage of the vaccine. Within this context, the development of scalable production and purification processes of His-tagged recombinant fragment of PspA from clade 3 (rfPspA3) in Escherichia coli BL21(DE3) was proposed. Fed-batch production was performed using chemically defined medium with glucose or glycerol as carbon source. Although the use of glycerol led to lower acetate production, the concentration of cells were similar at the end of both fed-batches, reaching high cell density of E. coli (62 g dry cell weight/L), and the rfPspA3 production was higher with glucose (3.48 g/L) than with glycerol (2.97 g/L). A study of downstream process was also carried out, including cell disruption and clarification steps. Normally, the first chromatography step for purification of His-tagged proteins is metal affinity. However, the purification design using anion exchange followed by metal affinity gave better results for rfPspA3 than the opposite sequence. Performing this new design of chromatography steps, rfPspA3 was obtained with 95.5% and 75.9% purity, respectively, from glucose and glycerol culture. Finally, after cation exchange chromatography, rfPspA3 purity reached 96.5% and 90.6%, respectively, from glucose and glycerol culture, and the protein was shown to have the expected alpha-helix secondary structure.

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“…(2)式可以用来判断蛋白与固定相间的作用力性质, 如果用溶质的 lg I 对 Z 作图有很好的线性关系就表明溶质与固定相之间只存在非选择性作用力, 反之则存在选择性作用力 [25] . 用表 2 中 5 种蛋白的 lg I 对 Z 作图, 结果见图 5. lg I 对 Z 作图有一定的线性关系, 但线性关系较差, R 2 只有 0.9658, 而且所得到的 J 值 0.592 与理论值 1.74 [27] 也相差甚远, 这表明在疏水模式下蛋白与固定相的作用除了非选择性疏水作用力以外还有其他选择性作用力存在, 根据固定相配基性质推断, 这种影响蛋白在疏水模式下保留顺序的选择性作用力应该就是氢键作用力. …”

Section: Z 和 Lg I 值对蛋白保留研究unclassified

Selectivity Research of Glutathione Bonded Column for Proteins

Ke¹,

Sun²,

Zhang³

et al. 2012

Acta Chim. Sinica

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The multifunctional stationary phase with the properties of weak cation exchange (WCX), hydrophobic and hydrogen bonding interaction was synthesized by bonded the glutathione (GSH) to the silica surface. Five standard proteins myoglobin (Myo), ribonuclease (RNase), cytochrome (Cyt-C), α-Chymotrypsin (α-Chy) and lysozyme (Lys) were selected to evaluate the column efficiency. The experiments show that the stationary phase has the good separation performance for proteins both in hydrophobic interaction chromatography (HIC) and ion exchange chromatography (IEC). More importantly, the proteins separation in both HIC and IEC mode can be accomplished only by a single column and a kind of mobile phase. In order to compare the resolution of multifunctional column with other column, two popular commercial columns of TSKgel Ether-5PW in HIC mode and TSKgel CM-5PW in IEC mode were tested under optimum condition. It was found that the multifunctional column has a good resolution both in IEC and HIC mode compared with commercial TSKgel columns. The eluted order of five proteins were the same on multifunctional and TSKgel CM-5PW column in IEC mode, but there is a big different between multifunctional column and TSKgel Ether-5PW column in HIC mode, the eluted order of Myo and RNase as well as α-Chy and Lys are changed completely. This indicates that the selectivity of the multifunctional column is significantly better than that of the TSKgel columns. The chromatographic retention behaviors and mechanisms for proteins in glutathione bonded column were studied. The results demonstrate that there are two retention mechanisms for a basic protein, at a low salt concentration, the retention time of proteins is decreased with the increasing of salt concentration and the proteins were separated by electrostatic force, while in high salt concentration the retention time of proteins is increased with the increasing of salt concentration and proteins were separated by hydrogen bonding interaction force. This multi-interaction forces retention mode can effectively improve the selectivity of HPLC column. The research provides a new solution for high performance separation of proteins, peptides and amino acids.

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Liquid chromatography of recombinant proteins and protein drugs

Cited by 62 publications

References 158 publications

HIC resolution of an IgG1 with an oxidized Trp in a complementarity determining region

HIC resolution of an IgG1 with an oxidized Trp in a complementarity determining region

Development of production and purification processes of recombinant fragment of pneumococcal surface protein A in Escherichia coli using different carbon sources and chromatography sequences

Selectivity Research of Glutathione Bonded Column for Proteins

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